JP6968054B2 - Compositions and Methods for Cancers Expressing PDE3A or SLFN12 - Google Patents

Description

Cross-reference to related applications This application claims the priority and interests of US Provisional Patent Application No. 62/204875 filed August 13, 2015, of which the entire disclosure is incorporated herein by reference. be.

Federally Funded R & D Description The invention was made with government support under grant number 3U54HG005032 awarded by the National Institutes of Health. Government has certain rights in the present invention.

Cancer kills more than 550,000 people in the United States and more than 8 million people worldwide each year. New drugs, including small molecules, molecules that affect tissue-specific growth requirements, and immunomodulators, have been shown to benefit a subset of patients whose cancer has unique genomic mutations or other characteristics. .. Unfortunately, many cancer patients are left without effective treatment options.

One approach to identify new anti-cancer agents is phenotypic screening to discover novel small molecules that show strong selectivity among cancer cell lines, followed by cell characteristics associated with drug responses. Chemogenomics for identification. In the 1990s, Weinstein and colleagues demonstrated that cytotoxic profiles of compounds could be used to identify cellular properties such as gene expression profiles and DNA copy counts that correlate with drug susceptibility. In recent years, automated high-throughput chemical susceptibility testing of large groups of cell lines combined with comprehensive genomic and phenotypic characterization of cell lines has the ability to characterize cancer cell lines that mediate responses to small molecules. It is increasing greatly. Phenotypic observations of small molecule susceptibility are similar to SLFN11 expression in irinotecan-sensitive cancer cell lines and EWS-FLI1 rearrangement in PARP inhibitor-sensitive cancer cell lines, respectively. May be associated with somatic changes.

Methods of characterizing malignant tumors at the molecular level are useful for stratifying patients and thereby promptly leading them to effective therapies. There is an urgent need for improved methods to characterize the reactivity of subjects with cancer.

As described below, the invention is a phosphodiesterase 3A (PDE3A) modulator (eg, by detecting co-expression of PDE3A and Schlafen 12 (SLFN12) polynucleotides or polypeptides in cancer cells derived from a patient. , 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one, zardaverin and anagrelide) -sensitive cancers (eg, black) Tumor, adenocarcinoma, lung cancer, cervical cancer, liver cancer, endometrial cancer, lung cancer, hematopoietic / lymph cancer, ovarian cancer, cervical cancer, soft tissue sarcoma, smooth muscle tumor, urinary tract It features a method of identifying patients with cancer (pancreatic cancer, thyroid cancer, kidney cancer, glioblastoma or breast cancer).

In one embodiment, the invention provides a method of killing or reducing the survival of cancer cells selected as responsive to a phosphodiesterase 3A (PDE3A) modulator. This method is a step in contacting cells with a PDE3A modulator and is selected as having an increase in PDE3A and / or Schlafen12 (SLFN12) polypeptide or polynucleotide relative to the reference, thereby reducing cancer cell survival. Includes steps to make. In another embodiment, the invention provides a method of reducing cancer cell growth in a subject preselected as having cancer that is responsive to a PDE3A modulator. This method is a step of administering a PDE3A modulator to a subject and is preselected by the subject detecting an increase in PDE3A and / or SLFN12 polypeptide or polynucleotide levels relative to the reference, thereby causing cancer cell proliferation in the subject. Includes steps to reduce. In one embodiment, the subject is preselected by detecting an increase in PDE3A and / or SLFN12 polypeptide or polynucleotide levels. In some embodiments, the PDE3A modulator is 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP), zardaverin and anagrelide. It is selected from the group consisting of.

In another embodiment, the invention provides a method of identifying a subject with cancer that is responsive to PDE3A regulation. This method comprises the step of detecting an increase in PDE3A and / or SLFN12 polypeptide or polynucleotide levels in a subject's biological sample relative to a reference, thereby identifying the subject as responsive to PDE3A regulation. In one embodiment, increased levels of PDE3A and SFLN1 polypeptides or polynucleotides are detected.

In some embodiments, increased levels of PDE3A and / or SLFN12 polypeptides are detected by a method selected from the group consisting of immunoblot, mass spectrometry and immunoprecipitation. In some other embodiments, increased levels of PDE3A and / or SLFN12 polynucleotides are detected by a method selected from the group consisting of quantitative PCR, Northern blots, microarrays, mass spectrometry and in situ hybridization. In some embodiments, the activity of PDE3A is reduced. The PDE3A modulator can be administered orally. The PDE3A modulator can be administered by intravenous injection.

In some embodiments, the cancer cells are melanoma, endometrial cancer, lung cancer, hematopoietic / lymphoma, ovarian cancer, cervical cancer, soft tissue sarcoma, smooth muscle tumor, urinary tract cancer, Pancreatic cancer, thyroid cancer, kidney cancer, glioblastoma or breast cancer. In some other embodiments, the cancer cells are not B cell proliferative cancers. In some embodiments, the cancer cells are not multiple myeloma. In some embodiments, the biological sample is a tissue sample.

In another aspect, the invention provides a kit for identifying a subject having cancer responsive to PDE3A regulation, comprising a capture reagent that binds to PDE3A and / or a capture reagent that binds to SLFN12. do. In one embodiment, the kit comprises a capture reagent that binds to PDE3A and a capture reagent that binds to SLFN12.

In yet another aspect, the invention provides a kit for reducing cancer cell growth in a subject preselected as responsive to a PDE3A modulator, comprising DNMDP, zardaverin and / or anagrelide. do.

The present invention treats subjects with cancer identified as responsive to treatment with a PDE3A modulator by detecting co-expression of PDE3A and / or Schlafen12 (SLFN12) polynucleotides or polypeptides of cancer. Provide a method. The compositions and articles defined by the present invention have been isolated or manufactured in connection with the examples provided below. Other features and advantages of the invention will become apparent from the detailed description and claims.

Definitions Unless otherwise defined, all technical and scientific terms used herein have meaning generally understood by one of ordinary skill in the art to which the invention belongs. The following references provide those skilled in the art with many general definitions of terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition 1994); The Cambridge Dictionary of Science and Technology. (Walker ed., 1988); The Glossary of Genetics, 5th Edition, R.M. Rieger et al. (E.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the following meanings to them, unless otherwise specified.

。 "Anagrelide" (IUPAC name 6,7-dichloro-1,5-dihydroimidazole (2,1-b) quinazoline-2 (3H) -on) means a small molecule phosphodiesterase inhibitor having the following structure: :

..

。 "Syrostamide" (IUPAC name N-cyclohexyl-N-methyl-4-[(2-oxo-1-H-quinoline-6-yl) oxy] butaneamide) means a small molecule inhibitor having the following structure:

..

。 "Cilostazol" (IUPAC name 6- [4- (1-cyclohexyl-1H-tetrazol-5-yl) butoxy] -3,4-dihydro-2 (1H) -quinolinone) is a small molecule having the following structure. Means inhibitor:

..

。 "DNDMP" (IUPAC name 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -on) is a small molecule inhibitor with the following structure: Means agent:

..

。 "Forskolin" (IUPAC name (3R, 4aR, 5S, 6S, 6aS, 10S, 10aR, 10bS) -6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo- 3-vinyldodecahydro-1H-benzo [f] chromen-5-ylacetate) means a small molecule inhibitor having the following structure:

..

。 "Revosimendan" (IUPAC name (E) -2-cyano-1-methyl-3-(4- (4-methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-yl) phenyl) guanidine) ) Means a small molecule inhibitor having the following structure:

..

。 "Mylrinone" (IUPAC name: 2-methyl-6-oxo-1,6-dihydro-3,4'-bipyridine-5-carbonitrile) means a small molecule inhibitor having the following structure:

..

。 "Papaverine" (IUPAC name 1- (3,4-dimethoxybenzyl) -6,7-dimethoxyisoquinoline) means a small molecule inhibitor having the following structure:

..

。 "Siguazodan" (IUPAC name (E) -2-cyano-1-methyl-3- (4- (4-methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-yl) phenyl) guanidine ) Means a small molecule inhibitor having the following structure:

..

。 "Sildenafil" (IUPAC name 1- [4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1-H-pyrazolo [4,3-d] pyrimidin-5-yl)) Phenylsulfonyl] -4-methylpiperazine) means a small molecule inhibitor having the following structure:

..

。 "Trekincin" (IUPAC name: 9,10-dimethoxy-3-methyl-2- (2,4,6-trimethylphenyl) imino-6,7-dihydropyrimid [6,1-a] isoquinoline-4-one ) Means a small molecule inhibitor having the following structure:

..

。 "Vardenafil" (IUPAC name 4- [2-ethoxy-5- (4-ethylpiperazine-1-yl) sulfonyl-phenyl] -9-methyl-7-propyl-3,5,6,8-tetraazabicyclo [ 4.3.0] Nona-3,7,9-triene-2-on) means a small molecule inhibitor having the following structure:

..

。 "Zardaverin (IUPAC name 3- [4- (difluoromethoxy) -3-methoxyphenyl] -1H-pyridazine-6-on)" means a small molecule inhibitor having the following structure:

..

In some other embodiments, any one of the compounds cilostamide, cilostazol, DNDMP, levocimendan, milrinone, papaverine, sigazodan, sildenafil, trekincin, vardenafil and zardaverine is a small molecule phosphodiesterase inhibitor. In another embodiment forskolin can be used in the method of the invention.

"PDE3A polypeptide" is at least 85% identical to the sequence provided by NCBI reference number NP_000912.3, which catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Means a protein or fragment thereof having sex. A representative human full-length PDE3A amino acid sequence is provided below:
(SEQ ID NO: 3)

Three PDE3A isoforms, PDE3A1, PDE3A2, and PDE3A3 are known. PDE3A1 contains amino acids 146 to 1141 of the full length PDE3A amino acid sequence, PDE3A2 isoform 2 contains amino acids 299 to 1141 of the full length PDE3A amino acid sequence, and PDE3A3 contains amino acids 483 to 1141 of the full length PDE3A amino acid sequence.

By "PDE3A polynucleotide" is meant any nucleic acid molecule, including DNA and RNA encoding a PDE3A polypeptide or fragment thereof. A representative PDE3A nucleic acid sequence is provided at NCBI reference number: NM_000921.4:
1 gggggccact gggaattcag tgaagagggc accctatacc atggcagtgc ccggcgacgc
61 tgcacgagtc agggacaagc ccgtccacag tggggtgagt caagccccca cggcgggccg
121 ggactgccac catcgtgcgg accccgcatc gccgcgggac tcgggctgcc gtggctgctg
181 gggagacctg gtgctgcagc cgctccggag ctctcggaaa ctttcctccg cgctgtgcgc
241 gggctccctg tcctttctgc tggcgctgct ggtgaggctg gtccgcgggg aggtcggctg
301 tgacctggag cagtgtaagg aggcggcggc ggcggaggag gaggaagcag ccccgggagc
361 agaagggggc gtcttcccgg ggcctcgggg aggtgctccc gggggcggtg cgcggctcag
421 cccctggctg cagccctcgg cgctgctctt cagtctcctg tgtgccttct tctggatggg
481 cttgtacctc ctgcgcgccg gggtgcgcct gcctctggct gtcgcgctgc tggccgcctg
541 ctgcgggggg gaagcgctcg tccagattgg gctgggcgtc ggggaggatc acttactctc
601 actccccgcc gcgggggtgg tgctcagctg cttggccgcc gcgacatggc tggtgctgag
661 gctgaggctg ggcgtcctca tgatcgcctt gactagcgcg gtcaggaccg tgtccctcat
721 ttccttagag aggttcaagg tcgcctggag accttacctg gcgtacctgg ccggcgtgct
781 ggggatcctc ttggccaggt acgtggaaca aatcttgccg cagtccgcgg aggcggctcc
841 aagggagcat ttggggtccc agctgattgc tgggaccaag gaagatatcc cggtgtttaa
901 gaggaggagg cggtccagct ccgtcgtgtc cgccgagatg tccggctgca gcagcaagtc
961 ccatcggagg acctccctgc cctgtatacc gagggaacag ctcatggggc attcagaatg
1021 ggaccacaaa cgagggccaa gaggatcaca gtcttcagga accagtatta ctgtggacat
1081 cgccgtcatg ggcgaggccc acggcctcat taccgacctc ctggcagacc cttctcttcc
1141 accaaacgtg tgcacatcct tgagagccgt gagcaacttg ctcagcacac agctcacctt
1201 ccaggccatt cacaagccca gagtgaatcc cgtcacttcg ctcagtgaaa actatacctg
1261 ttctgactct gaagagagct ctgaaaaaga caagcttgct attccaaagc gcctgagaag
1321 gagtttgcct cctggcttgt tgagacgagt ttcttccact tggaccacca ccacctcggc
1381 cacaggtcta cccaccttgg agcctgcacc agtacggaga gaccgcagca ccagcatcaa
1441 actgcaggaa gcaccttcat ccagtcctga ttcttggaat aatccagtga tgatgaccct
1501 caccaaaagc agatccttta cttcatccta tgctatttct gcagctaacc atgtaaaggc
1561 taaaaagcaa agtcgaccag gtgccctcgc taaaatttca cctctttcat cgccctgctc
1621 ctcacctctc caagggactc ctgccagcag cctggtcagc aaaatttctg cagtgcagtt
1681 tccagaatct gctgacacaa ctgccaaaca aagcctaggt tctcacaggg ccttaactta
1741 cactcagagt gccccagacc tatcccctca aatcctgact ccacctgtta tatgtagcag
1801 ctgtggcaga ccatattccc aagggaatcc tgctgatgag cccctggaga gaagtggggt
1861 agccactcgg acaccaagta gaacagatga cactgctcaa gttacctctg attatgaaac
1921 caataacaac agtgacagca gtgacattgt acagaatgaa gatgaaacag agtgcctgag
1981 agagcctctg aggaaagcat cggcttgcag cacctatgct cctgagacca tgatgtttct
2041 ggacaaacca attcttgctc ccgaacctct tgtcatggat aacctggact caattatgga
2101 gcagctaaat acttggaatt ttccaatttt tgatttagtg gaaaatatag gaagaaaatg
2161 tggccgtatt cttagtcagg tatcttacag actttttgaa gacatgggcc tctttgaagc
2221 ttttaaaatt ccaattaggg aatttatgaa ttattttcat gctttggaga ttggatatag
2281 ggatattcct tatcataaca gaatccatgc cactgatgtt ttacatgctg tttggtatct
2341 tactacacag cctattccag gcctctcaac tgtgattaat gatcatggtt caaccagtga
2401 ttcagattct gacagtggat ttacacatgg acatatggga tatgtattct caaaaacgta
2461 taatgtgaca gatgataaat acggatgtct gtctgggaat atccctgcct tggagttgat
2521 ggcgctgtat gtggctgcag ccatgcacga ttatgatcat ccaggaagga ctaatgcttt
2581 cctggttgca actagtgctc ctcaggcggt gctatataac gatcgttcag ttttggagaa
2641 tcatcacgca gctgctgcat ggaatctttt catgtcccgg ccagagtata acttcttaat
2701 taaccttgac catgtggaat ttaagcattt ccgtttcctt gtcattgaag caattttggc
2761 cactgacctg aagaaacact ttgacttcgt agccaaattt aatggcaagg taaatgatga
2821 tgttggaata gattggacca atgaaaatga tcgtctactg gtttgtcaaa tgtgtataaa
2881 gttggctgat atcaatggtc cagctaaatg taaagaactc catcttcagt ggacagatgg
2941 tattgtcaat gaattttatg aacagggtga tgaagaggcc agccttggat tacccataag
3001 ccccttcatg gatcgttctg ctcctcagct ggccaacctt caggaatcct tcatctctca
3061 cattgtgggg cctctgtgca actcctatga ttcagcagga ctaatgcctg gaaaatgggt
3121 ggaagacagc gatgagtcag gagatactga tgacccagaa gaagaggagg aagaagcacc
3181 agcaccaaat gaagaggaaa cctgtgaaaa taatgaatct ccaaaaaaga agactttcaa
3241 aaggagaaaa atctactgcc aaataactca gcacctctta cagaaccaca agatgtggaa
3301 gaaagtcatt gaagaggagc aacggttggc aggcatagaa aatcaatccc tggaccagac
3361 ccctcagtcg cactcttcag aacagatcca ggctatcaag gaagaagaag aagagaaagg
3421 gaaaccaaga ggcgaggaga taccaaccca aaagccagac cagtgacaat ggatagaatg
3481 ggctgtgttt ccaaacagat tgacttgtca aagactctct tcaagccagc acaacattta
3541 gacacaacac tgtagaaatt tgagatgggc aaatggctat tgcattttgg gattct tcgc
3601 attttgtgtg tatattttta cagtgaggta cattgttaaa aactttttgc tcaaagaagc
3661 tttcacattg caacaccagc ttctaaggat tttttaagga gggaatatat atgtgtgtgt
3721 gtatataagc tcccacatag atacatgtaa aacatattca cacccatgca cgcacacaca
3781 tacacactga aggccacgat tgctggctcc acaatttagt aacatttata ttaagatata
3841 tatatagtgg tcactgtgat ataataaatc ataaaggaaa ccaaatcaca aaggagatgg
3901 tgtggcttag caaggaaaca gtgcaggaaa tgtaggttac caactaagca gcttttgctc
3961 ttagtactga gggatgaaag ttccagagca ttatttgaat tctgatacat cctgccaaca
4021 ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgaaaga gagacagaag
4081 ggaatggttt gagagggtgc ttgtgtgcat gtgtgtgcat atgtaaagag atttttgtgg
4141 tttaagtaac tcagaatagc tgtagcaaat gactgaatac atgtgaacaa acagaaggaa
4201 gttcactctg gagtgtcttt gggaggcagc cattccaaat gccctcctcc atttagcttc
4261 aataaagggc cttttgctga tggagggcac tcaagggctg ggtgagaggg ccacgtgttt
4321 ggtattacat tactgctatg caccacttga aggagctcta tcaccagcct caaacccgaa
4381 agactgaggc attttccagt ctacttgcct aatgaatgta taggaactgt ctatgagtat
4441 ggatgtcact caactaagat caaatcacca tttaagggga tggcattctt tatacctaaa
4501 cacctaagag ctgaagtcag gtcttttaat caggttagaa ttctaaatga tgccagagaa
4561 ggcttgggaa attgtacttc agcgtgatag cctgtgtctt cttaatttgc tgcaaaatat
4621 gtggtagaga aagaaaagga aacagaaaaa tcactctggg ttatatagca agagatgaag
4681 gagaatattt caacacaggg tttttgtgtt gacataggaa aagcctgatt cttggcaact
4741 gttgtagttt gtctttcagg ggtgaaggtc ccactgacaa cccctgttgt ggtgttccac
4801 acgctgtttg ttggggtagc ttccatcggc agtctggccc attgtcagtc atgcttcttc
4861 tggccgggga gattatagag agattgtttg aagattgggt tattattgaa agtctttttt
4921 tttgtttgtt ttgttttggt ttgtttgttt atctacactt gtttatgctg tgagccaaac
4981 ctctatttaa aaagttgata ctcactttca atattttatt tcatattatt atatatgtca
5041 tgatagttat cttgatgtaa atatgaagat ttttttgttt ctgtagatag taaactcttt
5101 ttttaaaaaa ggaaaaggga aacattttta taaagttata ttttaatcac catttttata
5161 cattgtagtt ctctccaagc ccagtaagag aatgatgatt catttgcatg gaggtcgatg
5221 gacaaccaat catctacctt ttctaattta aatgataatc tgatatagtt ttattgccag
5281 ttaaatgagg atgctgcaaa gcatgttttt tcactagtaa cttttgctaa ctgaatgaat
5341 tctgggtcca tatctcccag atgaaaaact gttaaccaat accatatttt atagttggtg
5401 tccatttctt tccaacactg tttgttatga ttcttccttg agtacttata tacagacctg
5461 ctcattatct aaacaatctt accttctaag taaaccttga ttgtgatttc cagtttttat
5521 tttctctgac gtagtagaaa ggaatgttta cattaaaaat acttttgttt ctcataaatg
5581 gatattgtac tccccccttt caaagcatta ttttacaata attcatggca ttttaaaaaa
5641 taaggcaaag ataatacgac aaaaaatata catggtttca aggcaaattc tccaataagt
5701 tggaaaatgt aaaaaggatc aagtggatgc agcctctacc taaataatta aaatatattt
5761 cagtatattt ctgaattaac accaggtctt cattatttag aacttactaa attgttttca
5821 ttttcttagt tttacctgtg tatctccatg tttgcaaaaa ttactataag tcaaattttg
5881 ccagtgaatt taactatttt tctttccttg caattaaggg gaaaaaagca tttatcttat
5941 cttctcatac cccttgcatc taagtactta gcaaagtcaa tattttccca ttttccaaat
6001 gcgtccatct ctaacataaa tattaattga acatagagct atgtttggag tgagtggact
6061 ggcaggacag ttggaagtcc atcacagtct attgacagtt tcatcaaagc tgtatagtcc
6121 aactagtggg gcagcttggc tactatggtg gaagtctcag caaactgcct ggttttgttt
6181 gtttgttttg ttttaaggta caggaaataa gaggaataat agtggccaaa gcaattagaa
6241 catcttcatt ccagaactgt gttcagcaat ccaggcagat tgatacattt ttctttaaaa
6301 ataaattgct attacagcta gacgtcaatt gggataaata aagggatgaa gatccactaa
6361 gtttgtgact ttcatacaca cccagtacat ctcaaaggat gctaagggac attttctgcc
6421 agtagagttc tccccctttt tggtgacagc aatattatta tgttcacatc taactccaga
6481 gcttacttcc tgtggtgcca atgtatttgt tgcaatttac tacattttta tatgagccta
6541 tttataggtg ccattaaact caggtctttc aaatgaaaga gtttctagcc cacttaggga
6601 aaaagataat tgtttagaaa accataaaat caatggtagg aaaagttgga actggttacc
6661 tggatgccat ggttctctgt taaataaagt aagagaccag gtgtattctg agtgtcatca
6721 gtgttatttt cagcatgcta ataaatgtct ttccggttat atatctatct aaattaacct
6781 ttaaaatatt ggtttccttg ataaaagcac cacttttgct tttgttagct gtaatatttt
6841 ttgtcattta gataagacct ggtttggctc tcaataaaag atgaagacag tagctctgta
6901 cagggatata tctatattag tcttcatctg atgaatgaag aaattttctc atattatgtt
6961 caagaaagta tttacttcct aaaaatagaa ttcccgattc tgtctatttt ggttgaatac
7021 cagaacaaat ctttccgttg caatcccagt aaaacgaaag aaaaggaata tcttacagac
7081 tgttcatatt agatgtatgt agactgttaa tttgcaattt ccccatattt cctgcctatc
7141 ttacccagat aactttcttt gaaggtaaaa gctgtgcaaa aggcatgaga ctcaggccta
7201 ctctttgttt aaatgatgga aaaatataaa ttattttcta agtaataaaa gtataaaaat
7261 tatcattata aataaagtct aaagtttgaa attattaatt taaaaaaaaa aaaaaaaaa
(SEQ ID NO: 4)

"Schlafen12 (SLFN12) polypeptide" is at least 85% amino acid sequence identity with the sequence provided by NCBI reference number NP_060512.3, which interacts with PDE3A when bound to anagrelide, zardaverin or DNMDP and related compounds. Means a protein or fragment thereof. A representative human SLFN12 amino acid sequence is provided below:
(SEQ ID NO: 5)

By "Schlafen 12 (SLFN12) polynucleotide" is meant any nucleic acid molecule, including DNA and RNA encoding an SLFN12 polypeptide or fragment thereof. A representative SLFN12 nucleic acid sequence is provided at NCBI reference number: NM_018042.4:
1 tttgtaactt cacttcagcc tcccattgat cgctttctgc aaccattcag actgatctcg
61 ggctcctatt tcatttacat tgtgtgcaca ccaagtaacc agtgggaaaa ctttagaggg
121 tacttaaacc ccagaaaatt ctgaaaccgg gctcttgagc cgctatcctc gggcctgctc
181 ccaccctgtg gagtgcactt tcgttttcaa taaatctctg cttttgttgc ttcattcttt
241 ccttgctttg tttgtgtgtt tgtccagttc tttgttcaac acgccaagaa cctggacact
301 cttcactggt aacatatttt ggcaagccaa ccaggagaaa agaatttctg cttggacact
361 gcatagctgc tgggaaaatg aacatcagtg ttgatttgga aacgaattat gccgagttgg
421 ttctagatgt gggaagagtc actcttggag agaacagtag gaaaaaaatg aaggattgta
481 aactgagaaa aaagcagaat gaaagtgtct cacgagctat gtgtgctctg ctcaattctg
541 gagggggagt gatcaaggct gaaattgaga atgaagacta tagttataca aaagatggaa
601 taggactaga tttggaaaat tcttttagta acattctgtt atttgttcct gagtacttag
661 acttcatgca gaatggtaac tactttctga tttttgtgaa gtcatggagc ttgaacacct
721 ctggtctgcg gattaccacc ttgagctcca atttgtacaa aagagatata acatctgcaa
781 aagtcatgaa tgccactgct gcactggagt tcctcaaaga catgaaaaag actagaggga
841 gattgtattt aagaccagaa ttgctggcaa agaggccctg tgttgatata caagaagaaa
901 ataacatgaa ggccttggcc ggggtttttt ttgatagaac agaacttgat cggaaagaaa
961 aattgacctt tactgaatcc acacatgttg aaattaaaaa cttctcgaca gaaaagttgt
1021 tacaacgaat taaagagatt ctccctcaat atgtttctgc atttgcaaat actgatggag
1081 gatatttgtt cattggttta aatgaagata aagaaataat tggctttaaa gcagagatga
1141 gtgacctcga tgacttagaa agagaaatcg aaaagtccat taggaagatg cctgtgcatc
1201 acttctgtat ggagaagaag aagataaatt attcatgcaa attccttgga gtatatgata
1261 aaggaagtct ttgtggatat gtctgtgcac tcagagtgga gcgcttctgc tgtgcagtgt
1321 ttgctaaaga gcctgattcc tggcatgtga aagataaccg tgtgatgcag ttgaccagga
1381 aggaatggat ccagttcatg gtggaggctg aaccaaaatt ttccagttca tatgaagagg
1441 tgatctctca aataaatacg tcattacctg ctccccacag ttggcctctt ttggaatggc
1501 aacggcagag acatcactgt ccagggctat caggaaggat aacgtatact ccagaaaacc
1561 tttgcagaaa actgttctta caacatgaag gacttaagca attaatatgt gaagaaatgg
1621 actctgtcag aaagggctca ctgatcttct ctaggagctg gtctgtggat ctgggcttgc
1681 aagagaacca caaagtcctc tgtgatgctc ttctgatttc ccaggacagt cctccagtcc
1741 tatacacctt ccacatggta caggatgagg agtttaaagg ctattctaca caaactg ccc
1801 taaccttaaa gcagaagctg gcaaaaattg gtggttacac taaaaaagtg tgtgtcatga
1861 caaagatctt ctacttgagc cctgaaggca tgacaagctg ccagtatgat ttaaggtcgc
1921 aagtaattta ccctgaatcc tactatttta caagaaggaa atacttgctg aaagcccttt
1981 ttaaagcctt aaagagactc aagtctctga gagaccagtt ttcctttgca gaaaatctat
2041 accagataat cggtatagat tgctttcaga agaatgataa aaagatgttt aaatcttgtc
2101 gaaggctcac ctgatggaaa atggactggg ctactgagat atttttcatt atatatttga
2161 taacattctc taattctgtg aaaatatttc tttgaaaact ttgcaagtta agcaacttaa
2221 tgtgatgttg gataattggg ttttgtctat tttcacttct ccctaaataa tcttcacaga
2281 tattgtttga gggatattag gaaaattaat ttgttaactc gtctgtgcac agtattattt
2341 actctgtctg tagttcctga ataaattttc ttccatgctt gaactgggaa aattgcaaca
2401 cttttattct taatgacaac agtgaaaatc tcccagcata tacctagaaa acaattataa
2461 cttacaaaag attatccttg atgaaactca gaatttccac agtgggaatg aataagaagg
2521 caaaactcat (SEQ ID NO: 6)

In some embodiments, the compound is an isomer. An "isomer" is a different compound having the same molecular formula. A "stereoisomer" is an isomer that differs only in the way the atoms are arranged in space. As used herein, the term "isomer" includes any and all geometric and stereoisomers. For example, "isomers", as they fall within the scope of the invention, are geometric double bond cis-and trans-isomers, also called E-isomers and Z-isomers; R- and S-enantiomers; diastereomers. Includes stereomers, (d) -isomers and (l) -isomers, racemic mixtures thereof; and other mixtures thereof.

Geometric isomers, single bond as described herein, may be represented by ----- shows a bond that may be a double or triple bond. Provided herein are various geometric isomers and mixtures thereof resulting from the arrangement of substituents around carbon-carbon double bonds or the arrangement of substituents around carbon rings. Substituents around carbon-carbon double bonds are designated as "Z" or "E" configurations, and the terms "Z" and "E" are used according to IUPAC standards. Unless otherwise specified, structures exhibiting double bonds include both "E" and "Z" isomers.

Alternatively, the substituents around the carbon-carbon double bond can be called "cis" or "trans", where "cis" represents the substituent on the same side of the double bond and "trans" is the double bond. Represents a substituent on the opposite side of. The arrangement of substituents around the carbocycle can also be referred to as "cis" or "trans". The term "cis" refers to substituents on the same side of the surface of the ring, and the term "trans" refers to substituents on the opposite side of the surface of the ring. A mixture of compounds in which substituents are located on both the same and opposite sides of the surface of the ring is called a "cis / trans".

The term "enantiomer" refers to a pair of stereoisomers that are mirror images that cannot be superimposed on each other. Atoms with an asymmetric set of substituents can give rise to enantiomers. A mixture of any proportion of pairs of enantiomers can be known as a "racemic" mixture. The term "(±)" is used to indicate a racemic mixture where appropriate. A "diastereoisomer" is a stereoisomer that has at least two asymmetric atoms but is not a mirror image of each other. Absolute stereochemistry is specified by the Cahn-Ingold Prelogue RS system. If the compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Divided compounds of unknown absolute configuration can be represented by (+) or (−) depending on the direction (right-handed or left-handed) of rotating the plane polarized light at the wavelength of the sodium D line. Since some of the compounds described herein contain one or more asymmetric centers, from an absolute stereochemical point of view at each asymmetric atom, (R)-or (S)-. It can give rise to enantiomers, diastereomers and other stereoisomeric forms that can be defined as. The chemicals, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures.

The optically active (R)-and (S) -isomers can be prepared, for example, with chiral synthons or chiral reagents, or can be split using conventional techniques. Enantiomers can be isolated from the racemic mixture by any method known to those of skill in the art including chiral high pressure liquid chromatography (HPLC), chiral salt formation and crystallization, or can be prepared by asymmetric synthesis. ..

Optical isomers can be obtained by partitioning the racemic mixture by conventional methods, such as the formation of diastereoisomer salts, treatment with optically active acids or bases. Examples of suitable acids are tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, ditor oil tartaric acid and camphorsulfonic acid. Separation of the mixture of diastereoisomers by crystallization, followed by liberation of optically active bases from these salts, separates the isomers. Another method involves the synthesis of a covalent diastereoisomer molecule by reacting the disclosed compound with an activated form of an optically pure acid or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation and then hydrolyzed to give the enantiomerically concentrated compound. The optically active compound can also be obtained using an active starting material. In some embodiments, these isomers can be in the form of free acids, free bases, esters or salts.

In certain embodiments, the compounds of the invention can be tautomers. As used herein, the term "tautomer" refers to at least one formal transfer of a hydrogen atom and at least one change in valence (eg, a single bond to a double bond, a triple bond). A type of isomer containing two or more tautomerizable compounds resulting from a single bond and vice versa. "Tautomerization" includes proton transfer or proton shift tautomerization, which is considered a subset of acid-base chemistry. "Proton transfer tautomerization" or "proton shift tautomerization" involves the transfer of protons with altered binding order. The exact ratio of tautomers depends on several factors, including temperature, solvent and pH. If tautomerization is possible (eg, in solution), the chemical equilibrium of the tautomer can be reached. Tautomerization (ie, a reaction that provides a pair of tautomers) can be catalyzed by an acid or base, or can occur with or without the action or presence of an external agent. Representative tautomerization includes, but is not limited to, keto-enol; amide-imide; lactam-lactim; enamine-imine; enamine- (different) enamine tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentan-2,4-dione and 4-hydroxypenta-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridine-4-ol and pyridine-4 (1H) -one tautomer.

All chiral, diastereomeric, racemic and geometric isomers of the structure are intended unless specific stereochemistry or isomer morphology is specifically indicated. All methods used to prepare the compounds of the invention and the intermediates produced therein are considered part of the invention. All tautomers of the compounds shown or described are also considered part of the invention.

"Drug" means any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.

By "improving" is meant reducing, suppressing, attenuating, reducing, stopping or stabilizing the onset or progression of a disease.

By "modification" is meant a change (increase or decrease) in the expression level or activity of a gene or polypeptide detected by standard technically known methods such as those described herein. As used herein, modifications are about 10% change in expression level, preferably about 25% change, more preferably about 40% change, most preferably about 50% or more change in expression level. including.

"Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide, but has certain biochemical modifications that enhance the function of the analog as compared to the native polypeptide. Such biochemical modifications could increase the protease resistance, membrane permeability or half-life of the analog, eg, without altering ligand binding. Analogs may include unnatural amino acids.

In the present disclosure, "comprises," "comprising," "containing," "having," and the like can have meaning attributable to US patent law and "include." Can mean "includes", "including", etc; "consisting essentially of" or "consists essentially" is also in US patent law. With the meaning attributed, the term is open-ended and allows the existence of more than the enumeration, unless the basic or new features of the enumeration are changed by the existence of more than the enumeration. However, embodiments of the prior art are excluded.

"Detecting" refers to identifying the presence, absence or quantity of an analyte to be detected. In certain embodiments, the analyte is a PDE3A or SLFN12 polypeptide.

"Disease" means any condition or disorder that impairs or interferes with the normal functioning of cells, tissues or organs. Examples of diseases include melanoma, adenocarcinoma, lung cancer, cervical cancer, liver cancer and breast cancer.

"Effective amount" means the amount of a compound described herein required to improve the symptoms of a disease as compared to an untreated patient. The effective amount of one or more active compounds used to carry out the invention for the therapeutic treatment of a disease will vary depending on the mode of administration, age, body weight and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and dosing regimen. Such quantities are called "effective" quantities. In one embodiment, the compound is DNMDP, zardaverin or anagrelide.

The present invention provides several targets useful in the development of highly specific drugs for treating the disorders characterized by the methods described herein. In addition, the methods of the invention provide easy means for identifying safe therapies for use in a subject. In addition, the methods of the invention provide a pathway for analyzing substantially any number of compounds for effects on the diseases described herein with high throughput, high sensitivity and low complexity.

"Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion is preferably at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90 of the total length of the reference nucleic acid molecule or polypeptide. Contains%. Fragments are about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300. , About 400, about 500, about 600, about 700, about 800, about 900 or about 1000 nucleotides or amino acids.

"Hybridization" means a hydrogen bond that can be a Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bond between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "marker" or "biomarker" is meant any protein or polynucleotide having an expression level or activity change (eg, at the protein or mRNA level) associated with a disease or disorder. In certain embodiments, the marker of the invention is PDE3A or SLFN12.

By "modulator" is meant any agent that binds to a polypeptide and alters the biological function or activity of that polypeptide. Modulators include, but are not limited to, agents (eg, "inhibitors") that reduce or eliminate the biological function or activity of the polypeptide. For example, modulators can inhibit the catalytic activity of polypeptides. Modulators include, but are not limited to, agents that increase or decrease the binding of the polypeptide to another agent. For example, the modulator can promote the binding of one polypeptide to another. In some embodiments, the modulator of the PDE3A polypeptide is DNMDP. In some other embodiments, the modulator of the PDE3A polypeptide is an anagrelide or zardaverin.

By "reference" is meant a standard or control state.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding the polypeptide of the invention or fragments thereof. Such nucleic acid molecules do not have to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" with an endogenous sequence can typically hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding the polypeptide of the invention or fragments thereof. Such nucleic acid molecules do not have to be 100% identical to the endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" with an endogenous sequence can typically hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means a pair that forms a double-stranded molecule with a complementary polynucleotide sequence (eg, a gene described herein) or a portion thereof under various stringency conditions. do. (See, for example, Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152: 399; Kimmel, A.R. (1987) Methods Enzymol. 152: 507).

For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM less than trisodium citrate, more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. .. Low stringency hybridization can be obtained in the absence of an organic solvent such as formamide, whereas high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Can be done. Stringent temperature conditions typically include temperatures of at least about 30 ° C, more preferably at least about 37 ° C, and most preferably at least about 42 ° C. It is well known to those of skill in the art to change additional parameters such as hybridization time, concentration of detergent such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA. Different levels of stringency are achieved by combining these different conditions as needed. In a preferred embodiment, hybridization is performed in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS at 30 ° C. In a more preferred embodiment, hybridization is performed at 37 ° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide and 100 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, hybridization is performed in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide and 200 μg / ml ssDNA at 42 ° C. Any useful variation of these conditions will be readily apparent to those of skill in the art.

In most applications, the washing steps that follow hybridization also change in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As mentioned above, wash stringency can be increased by lowering the salt concentration or by raising the temperature. For example, the stringent salt concentration in the wash step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step typically include temperatures of at least about 25 ° C, more preferably at least about 42 ° C, and even more preferably at least about 68 ° C. In a preferred embodiment, the washing step is performed at 25 ° C. in 30 mM NaCl, 3 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step is performed at 42 ° C. in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step is performed at 68 ° C. in 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. Further variations of these conditions will be readily apparent to those of skill in the art. Hybridization techniques are well known to those of skill in the art, eg Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et al. ( Current Protocols in Molecular Biology, Wiley Interscience, NY, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, NY); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. It is described in.

"Substantially identical" is either a reference amino acid sequence (eg, any one of the amino acid sequences described herein) or a nucleic acid sequence (eg, any one of the nucleic acid sequences described herein). ) Means a polypeptide or nucleic acid molecule that exhibits at least 50% identity. Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical to the sequence used for comparison at the amino acid level or nucleic acid. Is.

Sequence identity is typically sequence analysis software (eg, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 Sequence Analysis Software Package, BLAST, BESTFIT, GAP or PILEUP / PRETTY BOX. It is measured using the program). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. .. A typical approach for determining the degree of identity can use the BLAST program, which shows sequences in which the probability scores between ^e-3 and e- ^{100 are closely related.}

By "subject" is meant, but not limited to, human or non-human mammals, such as mammals including cows, horses, dogs, sheep or cats.

The range provided herein is understood to be an abbreviation for all values within the range. For example, the range from 1 to 50 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49 or 50 is understood to include any number, combination or subrange of numbers.

As used herein, terms such as "treat," "treat," and "treat" refer to reducing or ameliorating the disorders and / or symptoms associated therewith. Although not excluded, it will be recognized that treating a disorder or condition does not require the complete elimination of the disorder, condition or symptom associated therewith.

Unless otherwise specified or obvious from the context, the term "or" is understood to be inclusive as used herein. Unless otherwise specified or obvious from the context, the terms "a", "an" and "the" are understood to be singular or plural as used herein.

Unless otherwise specified or self-evident from the context, the term "about" as used herein is understood to be within the normal tolerance range in the art, eg, within two standard deviations of the mean. NS. Approximately are 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.1% of the specified values. It can be understood as within 05% or 0.01%. Unless otherwise apparent from the context, all numbers provided herein are modified by the term about.

The enumeration of the list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of listed groups. The enumeration of embodiments of variables or embodiments herein includes the embodiments as any single embodiment or in combination with any other embodiment or portion thereof.

Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

Identification of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP), a potent and selective cancer cell cell line injury agent And characterization, mean survival of TP53 mutant NCI-H1734 cells, a non-small cell lung cancer cell line, and TP53 wild-type A549 cells, another lung cancer cell line, 48 hours after treatment at a concentration of 10 μM. It is a scatter diagram of 1924 kinds of compounds showing. DNMDP is indicated by a large arrow. Other compounds that selectively kill NCI-H1734 cells are indicated by small arrows. The positive control staurosporine is indicated by a long arrow. Identification of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP), a potent and selective cancer cell cell injury agent And is a line graph showing a panel of cell lines characterized and treated with DNMDP at the indicated concentrations for 48 hours. Identification of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP), a potent and selective cancer cell cell injury agent And is a line graph showing HeLa cell lines characterized and treated with isolated enantiomers at the indicated concentrations of DNMDP for 48 hours. The (R) -enantiomer had an _{EC 50} that was 500-fold lower than the (S) -enantiomer. Identification of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP), a potent and selective cancer cell cell injury agent It is a diagram showing and characterization and showing the structure of (R) -DN MDP. It is a figure which shows that DNMDP selectively killed NCI-H1734 and did not affect the cell viability of A549. NCI-H1734 and A549 cell lines were treated with the indicated compounds and concentrations for 48 hours. (R) -6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (R) -DN MDP) and analog synthesis schemes are shown. It is a figure. The reaction conditions are as follows: (a) Ac ₂ O (91%); (b) 90% HNO ₃ , H ₂ SO ₄ , (19%); (c) NaOH, MeOH / H ₂ O, ( 100%), then CH ₃ CHO, NaBH (OAc) ₃ (7%); (d) (BrCH ₂ CH ₂ ) ₂ O, K ₂ CO ₃ , DMF, (46%); (e) CH ₃ CHO , NaBH ₃ CN, MeOH (82%). 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) supercritical fluid (SCF) chromatograph. From top to bottom: three chromatographs showing ES +, diode array, ES-trace), peak 1 (CRO separation). 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) supercritical fluid (SCF) chromatograph. From top to bottom: three chromatographs showing ES +, diode array, ES-trace), peak 2 (CRO separation). 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) supercritical fluid (SCF) chromatograph. From top to bottom: three chromatographs showing ES +, diode array, ES-trace) and synthesized (R) -DN MDP (5:95 ratio peak 1: 2 by uv). Phosphodiesterase 3A (PDE3A) expression correlated with susceptibility to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP), but PDE3A Inhibition of mediated cAMP hydrolysis was not correlated with cytotoxicity, and is a scatter plot showing the correlation between DNMDP susceptibility and expression of 18988 genes in the 766 genomic characterization cell line. Cell lines were treated for 72 hours at concentrations ranging from 66.4 μM to 2 nM in 2-fold serial dilutions. The Z-score of the Pearson correlation between PDE3A expression and susceptibility to DNMDP is 8.5. Phosphodiesterase 3A (PDE3A) expression correlated with susceptibility to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP), but PDE3A It is a scatter plot showing the results of the panel A cell line treated with 480 compounds, showing that inhibition of mediated cAMP hydrolysis did not correlate with cytotoxicity. DNMDP showed the best correlation between PDE3A expression and susceptibility. Phosphodiesterase 3A (PDE3A) expression correlated with susceptibility to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP), but PDE3A Scatter plots showing _{published PDE3 inhibitor IC 50} and EC ₅₀ values for HeLa cells treated for 48 hours with the indicated compounds up to 10 μM, showing that inhibition of mediated cAMP hydrolysis did not correlate with cytotoxicity. Is. The DN MDP IC ₅₀ concentration for PDE3A inhibition was determined in Figure 7B. It is a figure which shows the chemical structure of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP). It is a figure which shows the chemical structure of sigazodan. It is a figure which shows the chemical structure of levosimendan. In a graph showing the determination of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) phosphodiesterase 3A (PDE3A) in vitro IC _50. It is a diagram showing PDE3A in vitro inhibition by positive control trekinsin at the indicated concentrations (IC ₅₀ curve performed by Caliper). In a graph showing the determination of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) phosphodiesterase 3A (PDE3A) in vitro IC _50. There is a diagram showing PDE3A in vitro inhibition by DNMDP at the indicated concentrations (IC ₅₀ curve performed by Caliper). Induction of cAMP signaling causes cytotoxicity induced by 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP). It is a graph which shows that the expression type was not copied. Forskolin: FSK. FIG. 5 shows cAMP concentrations measured 1 hour after treatment with the indicated compounds and concentrations in HeLa cells. Induction of cAMP signaling causes cytotoxicity induced by 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP). It is a graph which shows that the expression type was not copied. Forskolin: FSK. FIG. 5 shows the viability of HeLa cells treated with the indicated compounds and concentrations for 48 hours. Non-lethal phosphodiesterase 3 (PDE3) inhibitors compete for PDE3A binding to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H). ) -On (DNMDP) -induced cell death rescued, showing survival of HeLa cells treated with 1600 bioactive compounds at a concentration of 20 μM in combination with 30 nM (EC70) DNMDP for 48 hours. It is a scatter diagram. Survival was calculated as a percentage of untreated DMSO controls. Non-lethal phosphodiesterase 3 (PDE3) inhibitors compete for PDE3A binding to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H). -On (DNMDP) -induced cell death rescued, line showing survival of HeLa cells treated with DNMDP for 48 hours in combination with the indicated concentrations of non-lethal PDE3 and pan-PDE inhibitors It is a graph. Non-lethal phosphodiesterase 3 (PDE3) inhibitors compete for PDE3A binding to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H). 200 μg of HeLa cell lysate using a solid-phase-linked DNMDP linker analog showing salvage of on (DNMDP) -induced cell death and having the same salvage properties as non-lethal PDE3 inhibitors. It is a figure which shows the SDS-PAGE gel which shows the result of the affinity purification performed with respect to. The indicated compounds were incubated with linker analogs. The affinity purified fraction was run on an SDS-PAGE gel and examined for PDE3A. Linker compound tert-butyl (R)-(2-(2-(2- (ethyl (4-methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-yl) phenyl) amino) ) Ethoxy) ethoxy) ethyl) carbamate (DNMDP) -2L structure and salvage phenotype are shown and the structure of DNMDP-2L is shown. Linker compound tert-butyl (R)-(2-(2-(2- (ethyl (4-methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-yl) phenyl) amino) ) Ethoxy) ethoxy) ethyl) carbamate (DNMDP) -2L shows the structure and salvage phenotype and is a graph showing the viability of HeLa cells treated with the indicated compounds and concentrations for 48 hours. Phosphodiesterase 3A (PDE3A) is not required in susceptible cell lines, but is required to relay cytotoxic signals and is shown in Western blots. HeLa cells were infected with the indicated guide RNA (sgRNA) for Cas9 and PDE3A. Western blots were examined for PDE3A at the time indicated. Phosphodiesterase 3A (PDE3A) was not required in susceptible cell lines, but showed that it was required to relay cytotoxic signals and was infected with the indicated sgRNA for 2 weeks to 1 μM 6− (4-(diethylamino)). ) -3-Nitrophenyl) -5-Methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP) is a bar graph showing the rescue rate of HeLa cells treated for 48 hours. Relief rates were normalized to Cas9-only controls. Phosphodiesterase 3A (PDE3A) was not required in susceptible cell lines, but showed that it was required to relay cytotoxic signals and infected the indicated sgRNA with various concentrations of 6- (4- (diethylamino)). )-3-Methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP) -on (DNMDP) -treated cells viability plot. Decreased levels of phosphodiesterase 3A (PDE3A) protein cause resistance to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP). Western blots and graphs showing that HeLa cells were treated with a combination of four different siRNAs targeting scrambled control siRNAs or PDE3A. Cells were lysed at the indicated time points and immunoblotted for PDE3A and actin. Decreased levels of phosphodiesterase 3A (PDE3A) protein cause resistance to 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP). It is a Western blot and a graph showing that, and is a line graph showing the viability of HeLa cells treated with DNMDP analog 3 at the indicated concentration for 48 hours. Phosphodiesterase 3A (PDE3A) immunoprecipitation in the presence of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) is novel It is shown that the interaction between SIRT7 and SLFN12 was clarified, and it is a schematic diagram of the affinity enrichment of PDE3A performed in HeLa cells, followed by quantitative proteomics. All cells were treated for 4 hours prior to lysis with 10 μM of the indicated compound. The presence of all compounds was maintained throughout the experiment, including the wash step. Phosphodiesterase 3A (PDE3A) immunoprecipitation in the presence of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP) is novel The anti-PDE3A immunoprecipitate in DMSO-treated HeLa cells was enriched compared to the anti-PDE3A immunoprecipitate in the presence of a blocking peptide specific for the PDE3A antibody, showing that the interaction between SIRT7 and SLFN12 was revealed. It is a scatter plot showing the log ₂ ratio of a protein (each point represents a protein). Phosphodiesterase 3A (PDE3A) immunoprecipitation in the presence of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP) is novel It is a scatter plot showing _{the log 2} ratio of the change of the protein bound to PDE3A in the presence of DNMDP vs. trekincin, showing the clarification of the interaction between SIRT7 and SLFN12. Each point represents the average of two iterations per condition for an individual protein. In all cases, the plotted data passed the Bland-Altman test with a 95% confidence interval for reproducibility. The results of repeated PDE3A protein interaction tests using PDE3A as a bait under various conditions are shown. Each scatter plot shows a _{log 2} ratio of two iterations for proteins enriched with PDE3A under various conditions to enrichment with PDE3A in the presence of blocking peptide. _{Each point represents the log 2} ratio of that particular protein, the medium gray points correspond to Benjamini-Hochberg's adjusted p-value <0.01, and the light gray points are reproduced within the 95% confidence interval. Represents a protein outside the Blandt-Altman test for sex. It is a figure which shows that the protein enrichment was achieved by the immunoprecipitation using anti-PDE3A. The results of repeated PDE3A protein interaction tests using PDE3A as a bait under various conditions are shown. Each scatter plot shows a _{log 2} ratio of two iterations for proteins enriched with PDE3A under various conditions to enrichment with PDE3A in the presence of blocking peptide. _{Each point represents the log 2} ratio of that particular protein, the medium gray points correspond to Benjamini-Hochberg's adjusted p-value <0.01, and the light gray points are reproduced within the 95% confidence interval. Represents a protein outside the Blandt-Altman test for sex. It is a figure which shows that protein enrichment was achieved by immunoprecipitation using anti-PDE3A in the presence of DNMDP. The results of repeated PDE3A protein interaction tests using PDE3A as a bait under various conditions are shown. Each scatter plot shows a _{log 2} ratio of two iterations for proteins enriched with PDE3A under various conditions to enrichment with PDE3A in the presence of blocking peptide. _{Each point represents the log 2} ratio of that particular protein, the medium gray points correspond to Benjamini-Hochberg's adjusted p-value <0.01, and the light gray points are reproduced within the 95% confidence interval. Represents a protein outside the Blandt-Altman test for sex. It is a figure which shows that protein enrichment was achieved by immunoprecipitation using anti-PDE3A in the presence of trekincin. Cell lines with dual expression of SLFN12 and PDE3A were shown to be significantly enriched relative to DNMDP sensitive cell lines and have the indicated susceptible cell line Encyclopedia of Cancer Cell Lines (CCLE) Database (Human Cancer). Detailed genetic characterization of a large group of cell lines) is a scatter diagram showing mRNA robust multichip mean (RMA) expression levels of PDE3A and SLFN12 (Barretina et al., Nature 483, 603-607, 2012). Twenty-one susceptible cell lines were classified into three groups of seven based on subcurve area (AUC) rank. It was shown that cell lines with dual expression of SLFN12 and PDE3A were significantly concentrated relative to DNMDP sensitive cell lines, and expression of both SLFN12 and PDE3A (RMA Log2> 5) was compared with other cell lines. It is a bar graph showing the result of Fisher's exact test for the DNMDP sensitivity of a high cell line. The upper half of the right bar shows the melanoma cell line. It is a scatter plot showing the mRNA RPKM + 1 expression values for PDE3A and SLFN12 from the RNA sequencing data, showing that the cell lines with dual expression of SLFN12 and PDE3A were significantly enriched with respect to the DNMDP sensitive cell line. .. Cell lines with dual expression of SLFN12 and PDE3A were shown to be significantly enriched relative to DNMDP-sensitive cell lines, showing changes in SLFN12 qPCR expression in GAPDH-normalized shSLFN12 transduced HeLa cells. It is a bar graph showing. HeLa showing that cell lines with dual expression of SLFN12 and PDE3A were significantly enriched relative to DNMDP sensitive cell lines, transduced with the indicated shRNA reagents and treated with the indicated concentrations of DNMDP for 72 hours. It is a plot showing the viability of cells. Scatter plots showing that SLFN12 expression was among the top genes that correlate with DNMDP sensitivity, showing the correlation between DNMDP sensitivity and the expression of 18988 genes in the 766 genomic characterization cell line. It is a figure. Cell lines were treated for 72 hours at concentrations ranging from 66.4 μM to 2 nM in 2-fold serial dilutions. Scatter plots showing that SLFN12 expression was among the top genes that correlate with DNMDP sensitivity, showing the correlation between DNMDP sensitivity and the expression of 18988 genes in the 766 genomic characterization cell line. It is a scatter plot. Expression levels were corrected for PDE3A expression as described above (Kim et al., Genetica 131, 151-156, 2007). Cell lines were treated for 72 hours at concentrations ranging from 66.4 μM to 2 nM in 2-fold serial dilutions. It is a plot showing that DNMDP induces apoptosis in HeLa cells and shows the viability of HeLa cells treated with DNMDP at the indicated concentration for 48 hours. Caspase-Glo represents caspase 3/7 activity that indicates induction of apoptosis. CellTiter-Glo reflects survival. FIG. 6 shows that DNMDP induces apoptosis in HeLa cells and shows an immunoblot. HeLa cells were treated with the indicated compounds and concentrations for 36 hours. HeLa cells were harvested and immunoblotted for PARP cleavage products showing apoptosis. It is a scatter plot of PDE3A mRNA expression and susceptibility to DNMDP of 766 cancer cell lines. FIG. 6 shows an immunoblot showing that DNMDP induces an interaction between PDE3A and SIRT7 and SLFN12 in HeLa cells. HeLa cells were transfected with the indicated plasmid and treated with the indicated compound at a final concentration of 10 μM for 4 hours. Endogenous PDE3A was immunoprecipitated and immunoblotted for V5 to identify novel interactions with SIRT7 and SLFN12 (top two panels). Immunoprecipitate inputs were immunoblotted for PDE3A and V5 (bottom two panels). V5-SLFN12 could not be detected in whole cytolysis. It is a figure which shows the immunoblot which shows the confirmation of the mass spectrometric result of this specification using an affinity reagent. FIG. 20 shows that DNMDP and (weakly) anagrelide induced complex formation of PDE3A and SFLN12, but not trekincin. A set of tables showing the loss of SLFN12 in cells that have acquired resistance to DNMDP. It is a plot showing the sensitization of DNMDP resistant cell lines by the expression of SLFN12 or the expression of SFLN12 and PDE3A. It is a scatter plot showing the susceptibility of leiomyosarcoma (LMS) to PDE3A regulation based on the expression level of SLFN12.

Table 1 shows susceptibility data for 766 cancer cell lines treated with DNMDP. Cell lines were treated for 72 hours at concentrations ranging from 66.4 μM to 2 nM in 2-fold serial dilutions.

Table 2 shows the results of a panel of 19 phosphodiesterase inhibitory reactions performed by Caliper. The DNMDP concentration was 100 nM.

Table 3 shows the RPKM values for SLFN12 and PDE3A expression in multiple healthy histological types.

Table 4 shows the susceptibility of leiomyosarcoma to DNMDP.

Table 5 shows the binding of DNMDP to PDE3A (677-1141).

The compositions and articles defined by the present invention have been isolated or manufactured in connection with the examples provided below. Other features and advantages of the invention will become apparent from the detailed description and claims.

As described below, the invention is treated with a phosphodiesterase 3A (PDE3A) modulator by detecting co-expression of PDE3A and Schlafen 12 (SLFN12) polypeptides or polynucleotides in cancer cells derived from a patient. Cancers that are sensitive to (eg, melanoma, endometrial cancer, lung cancer, hematopoietic / lymphoma, ovarian cancer, cervical cancer, soft tissue sarcoma, smooth muscle tumor, urinary tract cancer, pancreas It features an improved method of identifying patients with cancer (thyroid cancer, kidney cancer, sarcoma or breast cancer). The present invention is at least partially sensitive to phosphodiesterase 3A modulators such as 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one or DNMDP. Is based on the finding that it correlates with the expression of the phosphodiesterase 3A gene, PDE3A, in 766 cancer cell lines. A subset of PDE3A inhibitors, such as DNMDP, kill selected cancer cells, but others do not; these cell-saving PDE3A inhibitors block DNMDP-induced cytotoxicity instead. In addition, PDE3A depletion results in DNMDP resistance. Binding of DNMDP to PDE3A facilitates the interaction of PDE3A with sirtuin 7 (SIRT7) and Schlafen12 (SLFN12), suggesting neomorphological activity and co-expression of SLFN12 and PDE3A that correlates with DNMDP sensitivity. do. These results indicate that the PDE3A modulator is a promising cancer therapeutic agent, demonstrating the ability of chemogenomics in small molecule discovery and target identification.

Therefore, the present invention is a method for selecting a subject having cancer that responds to the PDE3A modulator, and detects co-expression of PDE3A and Schlafen12 (SLFN12) polypeptide or polynucleotide in cancer cells derived from the subject. Provides a method that includes steps to do.

PDE3A modulator
Identification of PDE3A modulators was performed in connection with phenotypic screening designed to identify cytotoxic small molecules in the mutant tp53 background. The chemogenomics approach complements a targeted drug development program consisting of extensive in vitro and in vivo target validation and can also be referred to as reverse chemogenomics (Zheng et al., Curr Issues Mol Biol 4, 33-43, 2002). Many US Food and Drug Administration (FDA) approved targeted therapies have thus been developed, including small molecule kinase inhibitors targeting carcinogenic somatic driver mutations (Moffat et al., Nat Rev Drug Discov 13, 588-602). , 2014). However, the discovery and development of targeted therapies is often hampered by limited knowledge of the target's biological function, its mechanism of action, and the available chemicals to selectively inhibit the target.

Phenotypic screening can discover new targets for cancer therapy whose specific molecular mechanism is often elucidated by future studies (Swinney et al., Nat Rev Drug Discov 10, 507-519, 2011). ). In recent years, two classes of anticancer agents found by unbiased phenotypic screening studies have been approved by the FDA. While lenalidomide and pomalidomide have been found to be E3-ligase modulators that alter the affinity of their targets and lead to the degradation of lineage-specific transcription factors (Kronke et al., Science 343, 301-305, 2014; Lu). Et al., Science 343, 305-309, 2014), romidepsin and vorinostat were later identified as histone deacetylase (HDAC) inhibitors (Moffat et al., Nat Rev Drug Discov 13, 588-602, 2014; Nakajima et al. , Exp. Cell Res. 241, 126-133, 1998, Marks et al., Nat Biotechnol 25, 84-90, 2007).

Synthetic lethal approaches such as olaparib treatment for BRCA1 / BRCA2 mutant cancers have proven effective, but tumor suppressor changes are a good target for phenotypic screening because they cannot be directly targeted by small molecules. .. Current findings indicate that the tp53 tumor suppressor gene is the most frequently mutated across human cancers, with somatic mutations detected in 36% of 4742 cancers that have undergone total exome sequencing. Despite many attempts, no compound has been identified that selectively kills tp53 mutant cells.

Phenotypic screening developed to identify small molecules that cause synthetic lethality in tp53-mutated cancer cells is a class that acts as a modulator of phosphodiesterase 3A (PDE3A), as described below herein. The lucky discovery of selective cytotoxic agents has been made possible. Cyclic nucleotide phosphodiesterase catalyzes the hydrolysis of the secondary messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and is important in many physiological processes. Several phosphodiesterase inhibitors have been approved for clinical treatment, including the PDE3 inhibitors milrinone, cilostazol and levosimendan for cardiovascular indications and inhibition of platelet coagulation, and the PDE3 inhibitor anagrelide for plateletemia. PDE5 inhibitors, such as vardenafil, are used for smooth muscle disorders, including erectile dysfunction and pulmonary arterial hypertension, and the PDE4 inhibitor roflumilast reduces exacerbations from chronic obstructive pulmonary disease (COPD).

Phosphodiesterase inhibitors act by direct inhibition or allosteric regulation of their targets; for example, structural analysis of PDE4 resulted in the design of PDE4D and PDE4B allosteric modulators (Burgin et al., Nat Biotechnol 28, 63-70, 2010; Gurney et al., Neurootherapeutics 12, 49-56, 2015). The data provided below indicate that the cancer cytotoxic phosphodiesterase modulator DNMDP is likely to act through a similar allosteric mechanism.

Therefore, the present invention provides a method for identifying a subject having a malignant tumor that may respond to PDE3A modulator treatment based on PDE3A and SLFN12 expression levels in a subject's biological sample containing cancer cells. In some embodiments, the PDE3A modulator is DN MDP. In some other embodiments, the PDE3A modulator is an anagrelide or zardaverin.

Compound Form and Salt The compounds of the invention include the compounds themselves, as well as their salts and their prodrugs, if applicable. Salts can be formed, for example, between anions and positively charged substituents (eg, aminos) on the compounds described herein. Suitable anions include chlorides, bromides, iodides, sulfates, nitrates, phosphates, citrates, methanesulphonates, trifluoroacetates and acetates. Similarly, salts can also be formed between cations and negatively charged substituents (eg, carboxylates) on the compounds described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion and ammonium cation (such as tetramethylammonium ion). _{Examples of prodrugs include C 1-6} alkyl esters of carboxylic acid groups that, when administered to a subject, can provide an active compound.

The pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by adding a pharmaceutically acceptable acid or base to a compound disclosed herein. Point to. As used herein, the phrase "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological point of view and does not adversely interact with the active ingredient.

Examples of suitable acid salts are acetate, adipate, alginate, asparagate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, citrus acid, cerebral sulfonic acid. Salt, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptate, glycolate, hemisulfate, heptaneate, hexaneate, hydrochloride, hydrobromic acid Salt, hydride, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalensulfonate, nicotinate, nitrate, palmoate, Pectinate, persulfate, 3-phenylpropionate, phosphate, picphosphate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylic acid Examples include salts and undecanoates. Other acids, such as oxalic acid, which are not pharmaceutically acceptable in themselves, for the preparation of salts of the invention useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Can be used. Salts derived from appropriate bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and N- (alkyl) ₄ ⁺ salts. The invention also envisions quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Such quaternization can result in water or oil-soluble or dispersible products. The salt form of any of the compounds of the formula herein can be an amino acid salt of a carboxyl group (eg, L-arginine, -lysine, -histidine salt).

A list of suitable salts, each incorporated herein by reference in its entirety, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa. , 1985, p. 1418; Journal of Pharmaceutical Science, 66, 2 (1977); and "Pharmaceutical Salts: Properties, Selection, and Use A Handbook; Wermuth, CG and Stahl, PH (eds.) Verlag Helvetica. Found in Chimica Acta, Zurich, 2002 [ISBN 3-906390-26-8].

The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound by conventional methods. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but in other respects the salt is equivalent to the parent form of the compound for the purposes of the present invention. be.

In addition to the salt form, the invention provides compounds in the prodrug form. The prodrugs of the compounds described herein are compounds that are chemically altered under physiological conditions to provide the compounds of the invention. In addition, prodrugs can be converted to compounds of the invention by chemical or biochemical methods in an in vitro environment. For example, the prodrug can be slowly converted to a compound of the invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because they can be easier to administer than the parent drug in some circumstances. These may be biologically available, for example, by oral administration rather than the parent drug. The prodrug may also have better solubility in the pharmacological composition than the parent drug. A wide variety of prodrug derivatives are known in the art, including those by hydrolytic cleavage or oxidative activation of prodrugs. An example of, but not limited to, a prodrug would be a compound of the invention that is administered as an ester (“prodrug”), which is then metabolically hydrolyzed to the active substance carboxylic acid. Further examples include peptidyl derivatives of the compounds of the invention.

The invention also includes various hydrate and solvate forms of the compound.

The compounds of the present invention may also contain an unnatural proportion of atomic isotopes in one or more of the atoms constituting such a compound. For example, the compound may be radiolabeled with a radioisotope such as ^{tritium (3} H), iodine-125 ( ¹²⁵ I) or carbon-14 ( ^{14 C).} All isotopic variants of the compounds of the invention, whether radioactive or not, are intended to be included within the scope of the invention.

Diagnosis The present invention features a diagnostic assay for characterization of cancer. In one embodiment, levels of PDE3A and / or Schlafen12 (SLFN12) polynucleotides or polypeptides are measured in a subject sample and used as an indicator of cancer responsive to treatment with a PDE3A modulator. Levels of PDE3A and / or Schlafen12 polynucleotides can be measured by standard methods such as quantitative PCR, Northern blots, microarrays, mass analysis and in situ hybridization. Standard methods can be used to measure the levels of PDE3A and / or Schlafen12 polypeptide in a biological sample from a tumor. Such methods include immunoassays, ELISAs, Western blotting, and radioimmunoassays using antibodies that bind to PDE3A and / or Schlafen12. Elevated levels of PDE3A and Schlafen12 polynucleotides or polypeptides relative to the criteria are considered positive indicators of cancer responsive to treatment with PDE3A modulators.

Types of biological samples
Levels of PDE3A and / or SLFN12 expression are measured in various types of biological samples in characterizing the responsiveness of the subject's malignancies to PDE3A modulator treatment. In one embodiment, the biological sample is a tumor sample.

PDE3A and / or SLFN12 expression is higher in samples obtained from subjects responsive to PDE3A modulator treatment than expression levels in non-reactive subjects. In another embodiment, PDE3A and / or SLFN12 is at least about 5, 10, 20 or 30-fold higher in subjects with malignant tumors than in healthy controls. The multiple change value is determined using any method known in the art. In one embodiment, changes are determined by calculating differences in the level of expression of PDE3A and / or SLFN12 in cancer cells versus the level present in non-reactive cancer cells or in the corresponding healthy control cells.

Treatment Choices As reported herein, subjects suffering from malignant tumors can be tested for PDE3A and / or SLFN12 expression during the course of treatment choices. Patients characterized by increased PDE3A and / or SLFN12 relative to reference levels are identified as responsive to PDE3A modulator treatment.

Kits The present invention provides kits for characterizing a subject's responsiveness or resistance to PDE3A modulator therapy.

Also provided herein are kits that can include therapeutic compositions containing, for example, an effective amount of PDE3A modulator in unit dosage form.

In one embodiment, the diagnostic kit of the invention provides reagents for measuring the relative expression of PDE3A and SLFN12. Such reagents include capture molecules (eg, antibodies that recognize PDE3A and SLFN12 polypeptides or nucleic acid probes that hybridize to PDE3A and SLFN12 polynucleotides).

In some embodiments, the kit is a sterile container containing a therapeutic or diagnostic composition, such as a box, ampoule, bottle, vial, tube, bag, pouch, blister pack or other known in the art. Includes containers that may be of suitable container form. Such containers may be made of plastic, glass, laminated paper, metal foil or other material suitable for holding pharmaceuticals.

In one embodiment, the kit of the invention comprises reagents for measuring PDE3A and / or SLFN12 levels. If desired, the kit includes instructions for measuring PDE3A and / or SLFN12 and / or instructions for administering the PDE3A modulator to a subject with a malignant tumor selected as responsive to PDE3A modulator treatment. Including further. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration to treat or prevent the malignant tumor or its symptoms; precautions; warnings; indications; contraindications. Overdose information; Side effects; Animal pharmacology; Clinical trials; and / or references. Instructions may be printed directly on the container (if any), as a label affixed to the container, or as another sheet, pamphlet, card or folder that is placed in or provided with the container.

Unless otherwise indicated, the practice of the present invention uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology within the understanding of those skilled in the art. do. Such techniques are described in "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology". "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); well documented in literature such as "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the invention and can therefore be considered in the fabrication and practice of the invention. Particularly useful techniques for a particular embodiment are discussed in the following sections.

The following examples are presented to those of skill in the art to provide full disclosure and description of the fabrication and use of assays, screening and therapeutic methods of the invention and are intended to limit the scope of the invention. Not.

Example Example 1. Identification of cell-selective cytotoxic small molecules Two lung adenocarcinoma cell lines, A549 and NCI-H1734, both carcinogenic KRAS mutations and shortened, to identify anticancer compounds with cell-selective cytotoxic activity. Unbiased chemical screening was performed on TP53 wild-type and variant (R273L), respectively, with type STK11 mutations. 1924 compounds were screened from a small molecule storage confirmation set in the molecular library of A549 and NCI-H1734 cell lines at a single concentration of 10 μM in a 384-well format in duplicate. As a surrogate for cell viability, ATP content was measured 48 hours after compound treatment.

The three compounds showed a selective reduction in cell viability of the NCI-H1734 cell line compared to the A549 cell line, showing a reduction of approximately 50% in the NCI-H1734 cell line, which is the A549 cell line. It is an absolute deviation of more than 4 from the median in the negative direction compared to the minimum change of the median absolute deviation less than 1 from the median in (Fig. 1A). By retesting the three compounds in a dose-response analysis, one compound, 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H). ) -On, ie DNMDP, was confirmed to be specifically toxic to the NCI-H1734 cell line (Fig. 2).

Further cell line testing with DNMDP revealed cell-selective cytotoxicity with _{EC 50} of 10-100 nM for two additional lung adenocarcinoma cell lines, NCI-H1563 and NCI-H2122, and HeLa cervical cancer cells. _{However, EC 50} was over 1 μM for A549, MCF7 and PC3 cells (Fig. 1B; Fig. 1C). Caspase activity was detected by caspase-sensitive luciferase assay and poly-ADP ribose polymerase (PARP) cleavage in HeLa cells after DNMDP treatment, indicating that susceptible cells undergo apoptosis after DNMDP exposure (FIGS. 17A-17B). To further characterize cell susceptibility to DNMDP, 766 genomically characterized cancer cell lines were screened for 72-hour DMNDP susceptibility at concentrations ranging from 66.4 μM to 2 nM in the 2-fold dilution step. From these cell lines, 22 cell lines were classified as susceptible, with a robust Z score lower than -4, in particular representing multiple lineages containing multiple melanoma cell lines (Table 1).

The DNMDP enantiomers were then separated by chiral supercritical fluid (SCF) chromatography. One enantiomer was 500 times more potent in HeLa cells than the other (Fig. 1C and Fig. 1D). (R) -Enantiomers were synthesized from commercially available starting materials (Fig. 3). This synthetic enantiomer had similar activity to the more potent isolated material and was identical by chiral SCF chromatography, confirming the stereochemistry of the active enantiomer (FIGS. 4A-4C). Two (R) -des-nitro analogs of DNMDP were synthesized and both tested similarly to (R) -DN MDP (Fig. 3). FIGS. 4A-4C are supercritical fluid (SCF) chromatographs of 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP). It is a figure which shows the graph (from top to bottom: ES +, diode array, ES-trace). Figure 4A shows peak 1 (CRO separation); Figure 4B shows peak 2 (CRO separation); Figure 4C shows synthesis (R) -DN MDP (5:95 ratio peak 1: 2 by uv).

Example 2. Identification of PDE3A as a putative target for DNMDP
Considering the strong inhibition of cell-selective growth by 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3- (2H) -one (DNMDP), its action The mechanism was investigated in more detail. Chemo of 766 test cell lines previously characterized for mutations, copy counts and gene expression characteristics as part of the Cancer Cell Line Encyclopedia (CCLE, Barretina et al., 2012) to determine molecular targets for DNMDP. Genomic analysis was performed to look for correlations between these genomic features and DNMDP susceptibility. Analysis of the Pearson correlation between DNMDP susceptibility across cell line sets and expression of individual genes showed a strong correlation with expression of the PDE3A gene encoding phosphodiesterase 3A (Fig. 5A). The correlation between DNMDP susceptibility and PDE3A expression is not perfect (Figure 18), and manual validation of all 766 cell lines is not logistically feasible, resulting in some errors due to the high throughput of the cell line susceptibility characteristics. May be introduced. In contrast, mutation and copy number characteristics did not correlate with DNMDP susceptibility. Conversely, in the 480 compounds tested, DNMDP sensitivity was most correlated with PDE3A expression (Fig. 5B), and cancer cell lines with high PDE3A expression were more clearly sensitive to DNMDP than other test compounds. Was shown to be. In contrast to the motivation for initial screening, there was no correlation between TP53 mutations or other measures of p53 function and DNMDP susceptibility.

Given these results and the clear structural similarity of DNMDP to known PDE3 inhibitors such as levosimendan and siguazodan (FIGS. 6A-6C), biochemistry of DNMDP to 19 phosphodiesterases representing 11 PDE superfamily. Analysis was carried out. At a concentration of 100 nM, DNMDP specifically inhibited both PDE3A and PDE3B, weakly inhibited PDE10, and had little or no detectable effect on other phosphodiesterases (Table 2).

All PDE3 inhibitors are cytotoxic similar to DNMDP due to the cellular correlation between PDE3A expression and DNMDP sensitivity, in vitro inhibition of PDE3A and PDE3B by DNMDP, and the structural similarity of DNMDP to known PDE3 inhibitors. It was analyzed whether it showed a profile. Surprisingly, the IC ₅₀ for in vitro enzymatic PDE3A inhibition, there was little correlation between HeLa cell cytotoxicity over a series of test compound (Figure 5C and FIGS. 7A and 7B). In fact, the potent PDE3 inhibitor trekincin (PDE3 IC ₅₀ = 0.25nM, Ruppert et al., Life Sci. 31, 2037-2043, 1982) did not affect HeLa cell viability in a detectable manner. Despite the differential effect on HeLa cell viability, the non-cytotoxic PDE3 inhibitor trekincin and the potent cytotoxic compound DNMDP had similar effects on intracellular cAMP levels in forskolin-treated HeLa cells (Fig. 8A). And Figure 8B). This result indicates that inhibition of cAMP and cGMP hydrolysis function of PDE3A was not sufficient for the cytotoxic activity of DNMDP.

Example 3. Target verification of PDE3A
6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (DNMDP) and some PDE3 inhibitors are HeLa and other DNMDP sensitive cells However, other PDE3 inhibitors do not affect cell viability, the complex relationship between phosphodiesterase 3A (PDE3A) inhibition and cell death has shown several possible interpretations, including: 1 ) Cell-damaging activity was PDE3-independent, and due to its action on different proteins through screening, 234 kinases did not find kinase inhibition by 10 μM DNMDP; 2) cytotoxic and non-cytotoxic PDE3. Inhibitors may bind to different sites within the protein and exert different activities; or 3) cytotoxic and non-cytotoxic PDE3 inhibitors bind to PDE3 active sites, but the protein steric May have different effects on locus and activity. Although this third possibility may not be expected, the allosteric modulator of PDE4 binds to the PDE4 active site and interacts with the upstream (UCR2) and downstream (CR3) regulatory domains, thereby specific inactive components. It has been shown to stabilize the formation (Burgin et al., Nat Biotechnol, Vol. 28, pp. 63-70, 2010). Most importantly, PDE4 competitive inhibitors and PDE4 allosteric modulators have similar IC ₅₀ of about cAMP hydrolysis in vitro, had different cell activity and safety profile in animal studies (Burgin et al., Nat Biotechnol 28, 63-70, 2010). PHARMAKON 1600 collection of 1600 bioactive compounds to assess whether PDE inhibitors or other small molecules compete with DNMDP (PHARMAKON 1600 is a unique collection of 1600 known drugs from the US and International Pharmacopoeia. Was screened to identify compounds that could rescue DNMDP-induced cell death. HeLa cells were co-treated with 30 nM DNMDP (EC ₇₀ concentration) and 20 μM each bioactive compound. Cell viability after 48 hours of treatment was assessed by ATP consumption as described above. The five most potent compounds that rescued DNMDP-induced cell death were all PDE inhibitors, and the three most potent compounds, levosimendan, milrinone, and cilostazol, were all selective PDE3 inhibitors (Figure). 9A).

Follow-up experiments confirmed that sirostamide, levocimendan, milrinone and several other non-cytotoxic selective PDE3 inhibitors can rescue DNMDP cytotoxicity in a dose-dependent manner (Fig. 9B). The most potent DNMDP competitor was trekincin, with an "RC ₅₀ " (concentration that achieved 50% relief) of less than 1 nM; in contrast, PDE5 inhibitors such as sildenafil and vardenafil, as well as the pan-PDE inhibitor Izublast. (Idubulast) and dipyridamole were not effective competitors up to 10 μM in this assay (Fig. 9B). This showed that non-cytotoxic PDE3 inhibitors and DNMDP compete for binding to the same molecular target that mediates the cytotoxic phenotype.

To identify the molecular target of DNMDP, affinity purification was performed using the (R) -des-nitro-DN MDP solid phase ligated linker analog (Fig. 10A) incubated with HeLa cytolysis. This linker analog has the same DNMDP cytotoxic rescue phenotype as the non-cytotoxic PDE3 inhibitor described above (FIG. 10B), which has also been shown to bind to the same molecular target. It competed for molecular targets by adding a separate enantiomer of excess trekincin or DNMDP, but here only the (R) -enantiomer was cytotoxic. Immunoblots for the affinity purified substance PDE3A showed that PDE3A actually binds to the linker analog. Binding of PDE3A to the linker analog was blocked by both trekincin and (R) -DNMDP, but not by the non-cytotoxic enantiomer (S) -DNMDP (Fig. 9C). Therefore, it was concluded that both trekincin and (R) -DNMDP interfere with the binding of PDE3A to the ligated DNMDP analog and both molecules bind directly to PDE3A.

Based on the observation that DNMDP-sensitive cells express high levels of PDE3A and DNMDP competes with non-cytotoxic inhibitors for PDE3A binding, DNMDP mediates its cytotoxic phenotype through interaction with PDE3A. It was hypothesized that PDE3A abundance was a direct cytodeterminant of DNMDP sensitivity. To substantiate this hypothesis, we tested the effect of lowering PDE3A levels on the response to DNMDP. A short palindromic sequence (CRISPR) -associated CAS9 enzyme with a clustered and regularly arranged, targeted by three guide RNAs (sgRNAs) that target three different sites at the PDE3A locus, completes PDE3A expression. Caused disappearance (Cong et al., Science 339, 819-823, 2013). Although sgRNA2 and sgRNA3 almost completely reduced PDE3A protein levels, sgRNA1 had a moderate effect on PDE3A expression (Fig. 11A). Importantly, both sgRNA2 and sgRNA3 provided significant relief of toxicity by the active cytotoxic DNMDP analog 3 (FIGS. 11A and 11B and 5A-5C). Both sgRNA2 and sgRNA3 provided a significant relief of toxicity by DNMDP (Fig. 11C). No changes in proliferation rate or morphology were observed in HeLa cells with reduced PDE3A expression, indicating that PDE3A is not required for cell survival. In an independent approach using siRNA smart pools containing four different siRNAs targeting PDE3A, PDE3A expression was reduced in HeLa cell lines with a maximum efficiency of 70% between 24-72 hours after transfection. HeLa cells treated with siPDE3A was more EC ₅₀ for DNMDP analogs as compared to the control siRNA conditions (FIGS. 12A and 12B). Although not constrained by theory, it was concluded that DNMDP cytotoxicity requires PDE3A and that DNMDP may regulate the function of PDE3A.

Example 4. Determining Phosphodiesterase 3A mechanism of action of DNMDP (PDE3A) to protein abundance 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydro-pyridazin -3 (2H) - one ( DNMDP) cytotoxic dependence has recently been observed for lenalidomide acting by new or higher morphological mechanisms by stabilizing the interaction between cereblon and the IKAROS family zinc fingers 1 (IKZF1) and IKZF3. It showed a similar possible mechanism (Kronke et al., Science 343, 301-305, 2014; Lu et al., Science 343, 305-309, 2014). Furthermore, PDE4 allosteric modulators have been shown to bind and stabilize "closed" protein conformations, which are not competitive inhibitors but have been independently shown to bind uniquely to the PDE4 partner protein DISC1. (Millar et al., Science 310, 1187-1191, 2005). We characterized protein complexes in which PDE3A was present under normal conditions and investigated how these complexes change when PDE3A binds to DNMDP or the non-cytotoxic PDE3 inhibitor trekincin. PDE3A and interacting proteins from Hela cells were immunoprecipitated in the presence of DNMDP and trekincin, subsequently labeled with isobaric stable isotope tags for relative abundance and quantified by mass spectrometry (iTRAQ). / MS, Fig. 13A). PDE3A immunoprecipitates from HeLa cells include protein phosphatase 2 subunits (PPP2CA, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2D), calcineurin (PPP3R1, PPP3CA, Beca et al., Circ. Res. 112, 289-297, 2013), 14 Concentrated for multiple protein phosphatase subunits, including -3-3 (YWHAB, YWHAQ, YWHAG, YWHAZ, Pozuelo Rubio et al., Biochem. J. 392, 163-172, 2005) and tubulin (TUBA1C, TUBA1B) family members. (Fig. 13B and Fig. 14A). In addition, PDE3A and PDE3B were found to be present in the same previously reported protein complex (Malovannaya et al., Cell 145, 787-799, 2011).

Binding of DNMDP altered the composition of co-immunoprecipitated interacting proteins with PDE3A. Proteins in which the PDE3A immunoprecipitate was specifically enriched after treatment with DNMDP contained sirtuin 7 (SIRT7) and Schlafen 12 (SLFN12) (FIGS. 13C and 14B). These proteins interacted specifically with PDE3A in the presence of DNMDP and were not observed in trekincin-treated controls, but were known to be PDE3B interactants, the abhydrolase domain-containing protein 15 (ABHD15, Chavez et al., Biochem. Biophys. Res. Communi. 342, 1218-1222, 2006) was enriched with immunoprecipitates from trekincin-treated cells (FIGS. 13C and 14C). The DNMDP-promoted interaction between PDE3A and both SIRT7 and SLFN12 was confirmed with affinity reagents. Immunoprecipitation of endogenous PDE3A in HeLa cells treated with DNMDP, rather than DMSO or trekincin, enhanced ectopic expression V5 tag SIRT7 and SLFN12 complex formation with PDE3A, as evidenced by co-immunoprecipitation. (Fig. 19). Figure 20 further shows that DNMDP and (weak) anagrelides induced complex formation of PDE3A and SFLN12, but not trekincin.

Similar to PDE3A, overexpression of SLFN12 appears to have a cytotoxic effect in DNMDP-sensitive cell lines, contributing to the difficulty of detecting SLFN12 in whole cell lysates.

Enhanced interaction of PDE3A with SIRT7 and SLFN12 indicated that one or more of these interacting proteins may contribute to DNMDP susceptibility. SIRT7 mRNA expression was relatively constant in all cells tested, but co-expression of SLFN12 and PDE3A mRNA showed a strong correlation with DNMDP sensitivity; almost all DNMDP-sensitive cell lines produced high levels of SLFN12. It was expressed (Figs. 15A to 15C). Importantly, nearly half of the susceptible cell lines expressing high levels of SLFN12 and PDE3A were found to be melanoma cell lines (Fig. 15B). SLFN12 expression alone is also one of the top genes that correlates with susceptibility to DNMDP, supporting the hypothesis that SLFN12 is functionally involved in DNMDP-induced cytotoxicity (Fig. 16A). Furthermore, when corrected for PDE3A development, SLFN12 expression was the gene most highly correlated with DNMDP sensitivity (Fig. 16B). To assess whether SLFN12 is required for the cytotoxic phenotype of DMNDP, we reduced SLFN12 mRNA expression by 60% in HeLa cells by knockdown with two shRNAs (Figure 15D). Similar to reduced PDE3A expression, reduced SLFN12 expression did not result in cytotoxicity and, in fact, reduced sensitivity to DNMDP (Fig. 15E). These results indicate that SLFN12, like PDE3A, is required for the cytotoxic phenotype of DMNDP. The characterization of normal expression of SLFN12 and PDE3A by the GTEX consortium (Pierson, E. et al., PLoS Comput. Biol. 11, e1004220 (2015)) shows low expression of SLFN12 in normal tissues, while both PDE3A and SLFN12. High consortium is rarely observed (Table 3). This may suggest that the targeted toxicity of DNMDP and related compounds may be potentially limited.

FIG. 21 shows the loss of SLFN12 in cells that acquired resistance to DNMDP. Cell lines that were initially sensitive to DNMDP were made resistant by continuous exposure to DNMDP and then analyzed by RNA-seq. One gene was downregulated in both HeLa and H2122: SLFN12. Therefore, decreased SLFN12 levels indicate that the cells became resistant to DNMDP and other PDE3A modulators.

FIG. 22 shows sensitization of DNMDP resistant cell lines by SLFN12 expression or SFLN12 and PDE3A expression. Expression of SLFN12 was sufficient to confer DNMDP sensitivity to A549 cells. Addition of PDE3A expression resulted in further sensitization.

Leiomyosarcoma is a malignant leiomyosarcoma. Patient tumor samples from leiomyosarcoma were analyzed for expression of PDE3A and SLFN12 to characterize the susceptibility of leiomyosarcoma (LMS) to DNMDP. Leiomyosarcoma appears to be sensitive to DNMDP due to the prevalence between high-purity TCGA samples expressing high levels of PDE3A and SLFN12 (Figure 23, Table 4). P value of association between biomarker expression and leiomyosarcoma: 0.0001.

Differential scanning calorimetry (DSF) was used to demonstrate the binding of DNMDP to the purified PDE3A catalytic domain PDE3A (677-1141). In this experiment, 5 μM hsPDE3A (640–1141) was incubated in the absence or presence of 100 μM compound, as shown in Table 5. Binding buffer: 20 mM Hepes pH 7.4, 100 μM TCEP, 1 mM MgCl ₂ , 150 mM NaCl.

Using chemogenomics, a novel class of compounds targeting cancer dependence by small molecule regulation of PDE3A (exemplified by DNMDP) was discovered. These compounds bind PDE3A exclusively to non-cytotoxic PDE3 inhibitors, exerting new or hypermorphic effects on the function of PDE3A, resulting in altered protein-protein interactions. rice field. One unique protein-interaction partner, SLFN12, is highly expressed in DNMDP-sensitive cell lines and exhibits a functional role in the pathways for cytotoxic signaling. As a result, DNMDP was selective and potent across a large group of cancer cell lines.

Here, novel cytotoxic compounds were identified with high selectivity and low nM potency for cancer cell lines across multiple lineages. Using the gene expression correlation of chemogenomics, PDE3A was identified as a putative target for this small molecule DNMDP. Interestingly, loss of PDE3A expression resulted in resistance to DNMDP. In addition, after PDE3A immunoprecipitation, the novelty of PDE3A during DNMDP binding, followed by isotopic stable isotope tags for relative abundance and quantification by mass spectrometry (iTRAQ / MS), probably by allosteric regulation of PDE3A function. SLFN12 and SIRT7 as protein-protein interaction partners have been identified. Importantly, SLFN12 expression was the most correlated gene with DNMDP sensitivity when corrected for PDE3A expression. Single-gene or multi-gene expression correlations have been shown to help elucidate the mechanism of action and associated signaling pathways of small molecules. We have identified new biochemical targets for cancer treatment that are unlikely to be discovered by target identification approaches such as loss-of-function screening or genomic analysis.

PDE3A belongs to the superfamily of phosphodiesterases and forms the PDE3 family together with PDE3B. The PDE3 family has dual substrate affinity and hydrolyzes both cAMP and cGMP. Expression of PDE3A is highest in cardiovascular, platelet, kidney and oocytes (Ahmad et al., Horm Metab Res 44, 776-785, 2012). Because PDE3A inhibition in platelets impairs activation and platelet coagulation, the clinical PDE3 inhibitor cilostazol has been developed to treat intermittent claudication (Bedenis et al., Cochrane Database Syst Rev 10, CD003748, 2014). Other PDE3 inhibitors such as milrinone, amrinone and levocimendan have been shown to treat congestive heart failure in which a combination of vasodilation and elevated cardiac cAMP levels increases myocardial contractility (Movsesian et al., Curr Opin Pharmacol). 11, 707-713, 2011). None of these clinical inhibitors were able to replicate the cytotoxic phenotype of DNMDP, indicating that cyclic nucleotide hydrolysis was not sufficient to induce cell death in DNMDP-sensitive cell lines. There is.

Interestingly, however, other PDE3 inhibitors such as zardaverin, anagrelide and quazinone have previously been reported to have cellular cytotoxic properties in selected numbers of cancer cell lines (Sun et al., PLoS). ONE 9, e90627, 2014; Fryknas et al., J Biomol Screen 11, 457-468, 2006). Consistent with this finding, other PDE3 and PDE4 inhibitors were found not to replicate the cytotoxic phenotype of Zardaverin, and retinoblastoma protein retinoblastoma 1 (RB1) expression desensitized Zeldavelin-sensitive cell lines. It was reported to be isolated from cell lines (Sun et al., PLoS ONE 9, e90627, 2014). This finding was in contrast to this data, for which no correlation was identified between the cytotoxic activity of DNMDP and the number of copies of RB1 or mRNA expression. Another PDE3 inhibitor, anagrelide, uniquely inhibited megakaryocyte differentiation and resulted in apoptosis. Other PDE3 inhibitors tested did not have this activity (Wang et al., Br. J. Pharmacol. 146, 32-332, 2005; Espassin, Y. et al., J. Thromb. Haemost. N / a- n / a, 2015, doi: 10.1111 / jth.12850). The reported effects of zardaverin on cell viability and anagrelide on megakaryocytic differentiation were hypothesized to be mediated through the same PDE3A regulation as described in this study.

Multiple PDE3 inhibitors have been shown to be competitive inhibitors and occupy the catalytic binding sites of cAMP and cGMP (Card et al., Structure 12, 2233-2247, 2004; Zhan et al., Mol. Pharmacol. 62, 514. ~ 520, 2002). In addition, zardaverin is co-crystallized in a complex with PDE4D that occupies the cAMP binding site and is modeled to bind to PDE3B in a similar manner (Lee et al., FEBS Lett. 530, 53-58, 2002). .. Given the structural similarity between DNMDP and zardaverin, and the fact that DNMDP inhibits both PDE3A and PDE3B, it was hypothesized that the binding mode of DNMDP is very similar to that of zardaverin. This indicates that, in addition to acting as a cAMP / cGMP competitive inhibitor, DNMDP allosterically induces the conformation responsible for its cytotoxic phenotype. Allosteric regulation of phosphodiesterases has previously been described for PDE4, where small molecules bind to the active site and at the same time interact with regulatory domains that cross the PDE4 active site. As a result, allosteric modulators stabilized protein conformations that have been shown to bind differentially to different PDE4 partner proteins (Burgin et al., Nat Biotechnol 28, 63-70, 2010).

Studies of proteins associated with PDE3A may reveal both their normal function and how PDE3A modulators such as DNMDP kill cancer cells. PDE3A is involved in carcinogenic viral transformation and interacts with the protein phosphatase 2 subunit that is mutated in human cancer (Nagao et al., Int. Symp. Princess Takamatsu Cancer Res. Fund 20, 177-184, 1989; Imielinski et al., Cell 150, 1107-1120, 2012; Lawrence et al., Nature 499, 214-218, 2013), showing the role of PDE3A in cancer cell signaling. Although these interactions were not induced by DNMDP binding, the importance of protein phosphatases in cancer biology would justify further research.

Enhanced interaction between PDE3A and SLFN12 promoted by binding of DNMDP to PDE3A, and the correlation between susceptibility to DNMDP and SLFN12 expression requires an understanding of the functional effects of PDE3A-SLFN12 interaction. It was strongly shown that it was. However, little is known at this time about the functional role of SLFN12 in human physiology and cancer biology. SLFN12 is part of the schlafen gene family, which differs significantly between humans and rodents. The major differences are due to rapid gene evolution and positive selection (Bustos et al., Gene 447, 1-11, 2009). Therefore, SLFN12 does not have a mouse ortholog and interferes with the study of SLFN12 in well-understood model organisms. A single publication of SLFN12 showed the regulation of prostate cancer cell lines after ectopic expression of SLFN12 (Kovalenko et al., J. Surg. Res. 190, 177-184, 2014). Further studies on the function of SLFN12 and its interaction with PDE3A could elucidate the mechanism of DNMDP cytotoxicity. Two observations showed that DNMDP acts as a new or higher form of PDE3A function: 1) DNMDP-sensitive cancer cell lines are independent of PDE3A expression for survival, rather PDE3A knockdown is DNMDP. It resulted in resistance; and 2) DNMDP induced or enhanced protein-protein interactions when bound to PDE3A. Lenalidomide was an example of a small molecule that acted as a new or higher form, not as an enzyme inhibitor. Lenalidemid regulated specific protein-protein interactions between celebrone ubiquitin ligase and the squid transcription factor, which were subsequently targeted for degradation (Kronke et al., Science 343, 301-305, 2014; Lu). Et al., Science 343, 305-309, 2014). By analogy, DNMDP could directly stabilize the PDE3A-SLFN12 interaction, or DNMDP could allosterically stabilize the PDE3 conformation that binds to SLFN12. Any of these mechanisms can result in new or higher morphological phenotypes. Further characterization of the neomorphic phenotype induced by DNMDP may facilitate the synthesis of small molecules that do not inhibit cyclic nucleotide hydrolysis by PDE3A. The toxicity profile of such small molecules should differ from the PDE3 inhibitors prescribed for cardiovascular indications.

This study reveals a previously unknown role of PDE3A in cancer maintenance, whose function is modified by a subset of PDE3 inhibitors and toxic to a subset of cancer cell lines. These data indicate that DNMDP and its analogs have higher-order or neomorphic effects on PDE3A that result in cytotoxicity demonstrated by the cells becoming less sensitive to DNMDP with reduced levels of cellular PDE3A. rice field. These observations are comparable to other reports of allosteric regulation of phosphodiesterase (Burgin et al., Nat Biotechnol 28, 63-70, 2010), indicating that DN MDP and analogs may have similar effects on PDE3A. ing. The exact mechanism of cell-selective cytotoxicity remains unknown so far, but further studies on novel interactions with SLFN12, and possibly SIRT7, may be informative.

In summary, the studies herein have discovered the cancer cell cytotoxic small molecule DNMDP using differential cytotoxic screening. Profiling of DNMDP in 766 genomically characterized cancer cell lines revealed stereospecific nanomolar efficacy in approximately 3% of the cell lines tested. A search for sensitive genomic features revealed that elevated PDE3A expression strongly correlates with the DNMDP response. DNMDP inhibited PDE3A and PDE3B and had little or no activity against other PDEs. Unexpectedly, however, most other PDE3A inhibitors tested did not phenotypically replicate DNMDP, including the potent and selective PDE3A inhibitor trekincin. Simultaneous treatment of DNMDP-sensitive cells with trekincin competes with the cancer cell cytotoxic activity of DNMDP, and knockout of PDE3A rescues susceptible cells from DNMDP-induced cytotoxicity and induces new morphological changes in PDE3A. It was assumed that PDE3A was required for cell killing. Mass spectrometry of PDE3A immunoprecipitates alone or in the presence of DNMDP or trekincin revealed differential binding of SLFN12 and SIRT7 only in the presence of DNMDP. Similar to PDE3A, SLFN12 expression levels are elevated in DNMDP-sensitive cell lines, knockdown of SLFN12 by shRNA reduces cell sensitivity to DNMDP, and DNMDP-induced complex formation of PDE3A and SLFN12 is a cancer cytotoxic expression. Shown to be important for the type. Therefore, the results herein show PDE3A modulators as candidate cancer therapeutics and demonstrate the ability of chemogenomics in small molecule discovery.

The above experiment was performed using the following methods and materials.

Compound library screening in NCI-H1734 and A549 cell lines
1500 NCI-H1734 or 1000 A549 cells were sown in 384-well plates in 40 μl RPMI supplemented with 10% fetal bovine serum and 1% Pen / Strep. After 24 hours of plating, a library of 1924 small molecule compounds was added at a concentration of 10 μM. Staurosporine was used as a positive control for cytotoxicity at a concentration of 10 μM and DMSO was used as a negative control at a concentration of 1%. All compounds were incubated with the small molecules shown for 48 hours. After 48 hours, the 384-well plate was removed from the incubator and allowed to cool to room temperature for 20 minutes. Cell viability was assessed by adding 40 μl of 25% CELLTITERGLO® (Promega) in PBS using THERMO COMBI ™ or a multi-channel pipette and incubating for 10 minutes. The emission signal was read using Perkin-Elmer EnVision. Survival was calculated by normalizing to DMSO controls.

Compound susceptibility testing in cell lines
1000 HeLa (DMEM), 1000 A549 (RPMI), 500 MCF-7 (DMEM), 4000 PC3 (F12-K), 1000 NCI-H2122 (RPMI) or 1500 NCI -H1563 (RPMI) cells were sown in 384 well plates in 40 μl of corresponding growth medium supplemented with 10% fetal bovine serum. After 24 hours of plating, the indicated compounds were added at the indicated concentrations and incubated for 48 hours. Cell viability was assessed as described in Compound Library Screening for NCI-H1734 and A549 cell lines.

Caspase activity in HeLa cells
1000 HeLa cells were sown in 384-well plates in 40 μl of corresponding growth medium supplemented with 10% fetal bovine serum. After 24 hours of plating, the indicated compounds were added at the indicated concentrations and incubated for 48 hours. Promega Caspase-Glo was added according to the manufacturer's recommendations and luminescence was measured as described in the compound library screening for NCI-H1734 and A549 cell lines.

Large-scale cell line survival measurement
The susceptibility of 777 cancer cell lines (CCL) drawn from 23 different strains to DNMDP was measured. Each cell line was sown in the preferred medium in 1536 plates of white opacity at a density of 500 cells / well. After overnight incubation, DNMDP was added by acoustic transfer in double steps at 16 concentrations ranging from 66.4 μM to 2 nM (Labcyte Echo 555, Labcyte Inc., Sunnyvale, CA). After 72 hours of treatment, cellular ATP levels were measured using a ViewLux Microplate imager (PerkinElmer, Waltham, MA) as a substitute for viability according to the manufacturer's protocol (CELLTITERGLO®, Promega Corporation, Madison, WI). Normalized to background (medium only) and vehicle (DMSO) treated control wells.

Concentration response curves were fitted using a non-linear fit to a 2- or 3-parameter sigmoid function over all 16 concentrations with a low concentration asymptote set to the DMSO normalized value to provide an optimal 8-point dose curve over the range of compound susceptibility. Identified. Area under the 8-point dose curve (AUC) was calculated by numerical integration as a susceptibility metric for further analysis. Similar sensitivity measurements have been obtained for a collection of 480 other compounds that enable analysis to identify cell lines that respond uniquely to DNMDP (Broad Institute Cancer Therapeutics Response Portal, Inheritance of Human Cancer Cell Lines). See Database for Comprehensive Confirmation of Relationships Between Target and Systematic Characteristics and Small Molecule Sensitivity for a Complete List of Compounds).

Correlation between susceptibility measurement and basal gene expression
Gene-centric Robust Multichip Mean (RMA) -normalized basal mRNA gene expression data measured with the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array, CCLE, a large group of human cancer cells Detailed genetic characterization; downloaded from Barretina et al., Nature 483, 603-607, 2012). Pearson correlation coefficients were calculated between gene expression (18988 transcripts) and subcurve area (AUC) over 760 overlapping CCLs. For comparison between small molecules exposed to different numbers of CCLs, Fisher's transformation was used to transform the correlation coefficient.

Chemical Experiment Method General Details All reactions were carried out in a nitrogen (N2) atmosphere. All reagents and solvents were purchased from commercial suppliers and used as received. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (300 or 400 MHz ¹ H, 75 or 101 ^{MHz 13} C) spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) relative to NMR solvents. The data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quadruple, m = multiplet; one or more Coupling constant (Hz). Flash chromatography was performed on Teledyne Isco Combiflash Rf using 40-60 μm silica gel (60 Å mesh). Waters 2795 Separation Module and 3100 Mass Detector Using a Waters Symmetry C18 Column (3.5 μm, 4.6X100 mm) with a gradient of 0-100% CH3CN in water for 2.5 minutes with constant 0.1% formic acid. In, tandem liquid chromatography / mass spectrometry (LC / MS) was performed. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Elemental analysis was performed by Robertson Microlit Laboratories, Ledgewood NJ.

(R) -Synthesis of DNMDP

In 5 mL of acetic anhydride, (R) -6- (4-aminophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H) -one (A, Toronto Research Chemicals) 2.00 g (9.84 mmol) ) Was stirred for 1 hour, 30 mL of water was added, filtered, the solid was rinsed with water and dried to give 2.20 g (91%) of product B. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ10.92 (s, 1H), 10.13 (s, 1H), 7.74 (d, J = 8.9, 2H), 7.65 (d) , J = 8.8, 2H), 3.41-3.33 (m, 1H), 2.68 (dd, J = 6.8, 16.8, 1H), 2.23 (d, J = 16.7, 1H), 2.08 (s, 3H), 1.07 (d, J = 7.3, 3H). ¹³ C NMR (75 MHz, DMSO-d ₆ ) δ168.50, 166.27, 152.25, 140.27, 129.24, 126.24, 118.70, 33.47, 26.91, 24. 02, 15.87. HPLC: R _t 0.72 min, purity> 95%. MS: 246 (M + 1).

To 3.09 g (15.3 mmol) of B in which 30 mL of sulfuric acid was dissolved and cooled in an ice bath, 0.72 mL (15 mmol) of 90% nitric acid in 8 mL of sulfuric acid was added via an addition funnel for 10 minutes. After stirring for 1 hour, the mixture was poured onto ice. The yellow solid was filtered off, the water was rinsed with EtOAc several times, then dried and combined with the yellow solid. Chromatography with 40-60% EtOAc in hexanes gave 1.12 g (25%) of the product as a yellow solid, which was recrystallized from EtOAc. ^{1 1} H NMR (300 MHz, DMSO−d ₆ ) δ11.13 (s, 1H), 10.41 (s, 1H), 8.25 (d, J = 1.8, 1H), 8.07 (dd) , J = 1.8, 8.6, 1H), 7.71 (d, J = 8.6, 1H), 3.55-3.40 (m, 1H), 2.74 (dd, J = 6.9, 16.8, 1H), 2.27 (d, J = 16.8, 1H), 2.09 (s, 3H), 1.08 (d, J = 7.2, 3H). ¹³ C NMR (75 MHz, DMSO-d6) δ168.57, 166.31, 150.37, 142.19, 131.69, 131.32, 130.60, 125.07, 121.70, 33.30 , 26.81, 23.44, 15.64. TLC: Rf 0.25 (1: 1 EtOAc: hexane). HPLC: R _t 0.87 min, purity> 95%. MS: 291 (M + 1). HRMS Exact Mass (M + 1): 291.1088. Found: 291.1091

To 58 mg (0.20 mmol) of C dissolved in 10 mL of MeOH, a solution of 48 mg (1.2 mmol) of NaOH in 0.5 mL of water was added. After 1 hour, the reaction was concentrated, water was added, rinsed with EtOAc, dried and concentrated to give product D 48 mg (93%). ¹ H NMR (300 MHz, DMSO-d6) δ10.92 (s, 1H), 8.28 (d, J = 2.0, 1H), 7.87 (dd, J = 2.1, 9.0) , 1H), 7.76 (s, 2H), 7.06 (d, J = 9.0, 1H), 3.33 (s, 1H), 2.67 (dd, J = 6.8, 16) .8, 1H), 2.22 (d, J = 16.6, 1H), 1.06 (d, J = 7.3, 3H). ¹³ C NMR (75 MHz, DMSO-d ₆ ) δ166.25, 151.12, 146.69, 132.72, 129.80, 122.57, 122.19, 119.80, 33.43, 26. 70, 15.77. MS: 249 (M + 1).

In dimethylformamide (DMF) amine was dissolved in 0.5mL D35mg (0.14mmol), was added acetaldehyde 70 mg (1.6 mmol) and _{NaBH (OAc) 3 170mg (0.80mmol} ) and HOAc10μL a (0.2 mmol). After stirring for 3 hours, water and EtOAc were added, EtOAc was separated, dried, concentrated and chromatographed with 30-50% EtOAc in hexanes to isolate (R) -DN MDP 3 mg (7%). bottom. The synthesized material was identical to the racemic material purchased by ^{TLC, HPLC and 1 H NMR. 1} H NMR (300 MHz, CDCl ₃ ) δ8.58 (s, 1H), 8.04 (d, J = 2.3, 1H), 7.84 (dd, J = 2.3, 9.0, 1H), 7.11 (d, J = 9.0, 1H), 3.30-1.36 (m, 1H), 3.26 (q, J = 7.1, 4H), 2.71 ( dd, J = 6.8, 16.9, 1H), 2.48 (d, J = 17.0, 1H), 1.25 (d, J = 7.4, 3H), 1.16 (t) , J = 7.1, 6H). TLC: Rf 0.25 (1: 1 EtOAc: hexane). HPLC: R _t 1.27 min, purity> 95%. MS: 305 (M + 1). Exact Mass (M + 1): 305.1608 Found: 305.1616. ¹³ C NMR (75 MHz, CDCl ₃ , purchased material) δ166.28, 152.02, 145.24, 141.21, 129.77, 124.94, 123.94, 121.00, 46.10, 33 .80, 27.81, 16.24, 12.56.

The optical purity of (R) -DN MDP was determined using chiral SCF chromatography and comparison with commercially available racemates: Column: ChiralPak AS-H, 250 x 4.6 mm, 5 μm, Mobile Phase Modifier: 100. % Methanol, gradient: 5-50% methanol over 10 minutes, flow rate: 4 mL / min, back pressure: 100 bar, column temperature: 40 ° C. UV detection was 200-400 nm. Retention time of the separated isomers: 5.36 minutes, 6.64 minutes; (R) -DN MDP retention time, 6.60 minutes, 1:19 ratio enantiomers were detected.

2. To 200 mg (0.98 mmol) of A dissolved in 5 mL of MeOH, 87 mg (2.0 mmol) of acetaldehyde, 113uL (2.0 mmol) of HOAc and 124 mg (2.0 mmol) of NaBH ₃ CN were added, and the reaction was stirred overnight at room temperature. The next day, the same amount of reagent was added and the reaction was stirred for an additional 24 hours. The mixture is concentrated _{, partitioned into CH 2} Cl ₂ and water, CH ₂ Cl ₂ is separated, dried, concentrated and then chromatographed in 20-40% EtOAc in hexanes to give 210 mg of the product as a white solid ( 82%). ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.95 (s, 1H), 7.64 (d, J = 8.7, 2H), 6.66 (d, J = 8.7, 2H), 3 .37 (dd, J = 9.6, 16.4, 5H), 2.67 (dd, J = 6.5, 16.8, 1H), 2.43 (d, J = 16.8, 1H) ), 1.41-1.02 (m, 10H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ166.82, 154.55, 148.79, 127.32, 120.81, 111.08, 44.32, 33.92, 27.74, 16.37, 12.50. TLC: Rf 0.25 (1: 1 EtOAc: hexane). HPLC: R _t 1.05 min, purity> 95%. MS: 260 (M + 1). HRMS Exact Mass (M + 1): 260.1757. Found: 260.1764

3. 3. A mixture of 250 μL (2.00 mmol) of bis (2-bromoethyl) ether and 400 mg of K2CO3 was added to 200 mg (0.994 mmol) of A dissolved in 1 mL of dimethylformamide (DMF), and the mixture was stirred overnight at 60 ° C. The next day, 250 μL of bis (2-bromoethyl) ether and 170 mg of K2CO3 were further added. After 3 hours, EtOAc and water were added, the water was rinsed with EtOAc, and the combined EtOAc wash was dried and concentrated. Chromatography with 0-4% MeOH in CH2Cl2 gave 125 mg (46%) of product. ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.61 (s, 1H), 7.68 (d, J = 8.8, 2H), 6.92 (d, J = 8.8, 2H), 3 .99-3.76 (m, 4H), 3.44-3.31 (m, 1H), 3.29-3.22 (m, 4H), 2.70 (dd, J = 6.7, 16.8, 1H), 2.46 (d, J = 16.7, 1H), 1.24 (d, J = 7.3, 3H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ166.64, 154.05, 152.18, 127.10, 125.33, 114.73, 66.69, 48.33, 33.93, 27.94, 16.36. TLC: R _f 0.1 (1:50 MeOH: CH ₂ Cl ₂ ). HPLC: R _t 1.05 min, purity> 95%. MS: 274 (M + 1). HRMS: calcd. 274.1556 (M + 1); found 274.1552. Anal. Calcd. for C ₁₅ H ₁₉ N ₃ O ₂ : C, 65.91; H, 7.01; N, 15.37; Found. 65.81, H, 6.66, N, 15.26.

DNMDP-2L. Tert-Butyl 2- (2- (2-bromoethoxy) ethoxy) -ethylcarbamate 100 mg (Toronto Research Chemical, 0.32 mmol) and K in 130 mg (0.64 mmol) of A dissolved in 0.4 mL of dimethylformamide (DMF). ₂ CO ³ 90 mg (64 mmol) was added and the mixture was stirred at 60 ° C. overnight. After cooling, water was added and rinsed with EtOAc several times. The combined EtOAc layers were dried, concentrated and chromatographed with 50-70% EtOAc to give 81 mg (58%) of product. ¹ H NMR (300 MHz, CDCl ₃ ) δ9.06 (s, 1H), 7.59 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 8.8 Hz, 2H) , 5.15 (s, 1H), 4.53 (s, 1H), 3.72 (t, J = 5.2 Hz, 2H), 3.65 (s, 4H), 3.55 (t, J = 5.2 Hz, 2H), 3.32 (m, 5H), 2.67 (dd, J = 16.8, 6.7 Hz, 1H), 2.42 (d, J = 16.4) Hz, 1H), 1.44 (s, 9H), 1.22 (d, J = 7.4 Hz, 3H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ166.83, 155.99, 154.45, 149.64, 127.33, 123.24, 112.58, 79.28, 70.30, 70.26, 70.22, 69.45, 43.14, 40.39, 33.96, 28.43, 27.89, 16.40; HPLC: R _t 2.50 min (7.5 min run), purity> 95%. MS: 435 (M + 1). The product (0.19 mmol) was dissolved in 1 mL of MeOH and acetaldehyde (50 μL, 0.89 mmol), HOAc 10 μL (0.2 mmol) and NaBH ₃ CN 12 mg (0.19 mmol) were added to the solution. After 1 hour, dichloromethane ₃ (aqueous solution) and CH ₂ Cl ₂ were added, CH ₂ Cl ₂ was separated, and water was washed twice _{with CH 2} Cl _2. The combined CH ₂ Cl ₂ was dried, concentrated and chromatographed in 60-70% EtOAc in hexanes to give 71 mg of product as clear oil (82%). ¹ H NMR (400 MHz, CDCl ₃ ) δ8.91 (s, 1H), 7.63 (d, J = 8.9 Hz, 2H), 6.69 (d, J = 8.9 Hz, 2H) , 5.07 (s, 1H), 3.65 (t, J = 6.0 Hz, 2H), 3.61 (s, 4H), 3.55 (dt, J = 9.9, 5.5) Hz, 4H), 3.46 (q, J = 7.0 Hz, 2H), 3.38-3.22 (m, 3H), 2.67 (dd, J = 16.8, 6.7 Hz) , 1H), 2.43 (d, J = 16.7 Hz, 1H), 1.45 (s, 10H), 1.23 (d, J = 7.3 Hz, 3H), 1.18 (t) , J = 7.0 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ166.84,155.96, 154.46, 148.89, 127.35, 121.38, 111.28, 79.22, 70.68, 70.27, 70.24, 68.74, 49.95, 45.49, 40.32, 33.97, 28.43, 27.80, 16.43, 12.14. R _t 2.99 min (7.5 min run), purity> 95%. MS: 463 (M + 1).

Adsorption to resin
To a solution of DNMDP-2L 18 mg (0.04 mmol) in CH ₂ Cl ₂ 0.8 mL, add 0.2 mL of trifluoroacetic acid (TFA), stir the solution for 2 hours, concentrate, and dissolve in 0.5 mL of DMSO. bottom. To this was _{added Et 3} N 10 μL (0.07 mmol) and N, N'-discusin imidazole carbonate (DSC) 12 mg (0.05 mmol), and the solution was stirred overnight. Since LC analysis showed that the reaction was not complete, an additional 25 mg (0.1 mmol) of N, N'-discusin imidazole carbonate was added. LC analysis after 2 hours showed a DSC product: amine in a ratio of about 5: 1. A 1 mL sample of Affi-Gel 102 resin was rinsed 5 times in DMSO using a centrifuge and then suspended in 0.5 mL DMSO. 30 μL of DSC product solution and 25 μL of Et3N were added to the resin and the mixture was swirled. Two days later, LC analysis of the DMSO solution showed complete disappearance of the DCS adduct. Underrivatized amines were still present. DMSO was removed by centrifugation, decanted, the resin rinsed several times with DMSO and stored in PBS buffer.

Screening for bioactive agents to relieve DNMDP-induced cytotoxicity
1000 HeLa cells were sown in 384-well plates in 40 μl DMEM supplemented with 10% fetal bovine serum and 1% Pen / Strep. After 24 hours of plating, a compound library of 1600 bioactive molecules (Pharmacon) was added at a concentration of 20 μM. In parallel with the incubation of the bioactive compound, DNMDP was added to a final concentration of 30 nM and incubated for 48 hours. Cell viability was assessed as described in Compound Library Screening for NCI-H1734 and A549 cell lines.

Linker affinity purification and immunoblot for molecular targets of DNMDP
HeLa cells were washed with ice-cold PBS and then supplemented with EDTA-free protease inhibitor (Roche) and phosphatase inhibitor mixtures I and II (Calbiochem) NP-40 lysis buffer (150 mM NaCl, 10% glycerol, 50 mM). It was dissolved in Tris-Cl pH 8.0, 50 mM MgCl ₂ , 1% NP-40). Cytolysis was incubated on ice for at least 2 minutes, then centrifuged at 15700 xg at 4 ° C. for 10 minutes, after which the supernatant was quantified using the BCA protein assay kit (Pierce). A total of 200 μg of HeLa cytolysis was incubated with 3 μl of Affi-Gel102 resin (BioRad) bound to the affinity linker DNMDP-2L for 4 hours in a total volume of 400 μl. Prior to incubation, the indicated compounds were added to affinity purification at a final concentration of 10 μM. The sample was washed 3 times with lysis buffer containing the corresponding compound concentration of 10 μM. Proteins bound to the Affi-Gel 102 resin were reduced, denatured, separated using Tris-glycine gel (Novex) and transferred to nitrocellulose membranes using the iBlot transfer system (Novex). Membranes were incubated overnight at 4 ° C. with primary antibody to PDE3A (1: 1000, Bethyl). Incubation with secondary antibody (1: 20000, LI-COR Biosciences) for 2 hours at room temperature and subsequent detection (Odyssey Imaging System, LI-COR Biosciences) was performed according to the manufacturer's recommendations.

PARP-Cut immune blot
HeLa cells were treated with the indicated concentrations of DNMDP and staurosporine for 36 hours. HeLa cells were lysed and treated as described in the linker affinity purification and immunoblot of the molecular target of DNMDP. Membranes were incubated with antibody against PARP (1: 1000, Cell Signaling # 9532) and actin and then imaged as described in the linker affinity purification and immunoblot of the molecular target of DNMDP.

Targeting the PDE3A locus using CRISPR
CRISPR target sites were identified using the MIT CRISPR design tool (online MIT CRISPR design portal). For cloning of sgRNA, forward and reverse oligos were annealed, phosphorylated and ligated to BsmBI digestion pXPR_BRD001. The oligo sequences are as follows:

293 T cells with pXPR_BRD001, psPAX2 and pMD2 using calcium phosphate to produce lentivirus. G was introduced at the same time. Infected HeLa cells were selected with 2 μg / ml puromycin.

Decreased PDE3A expression with siRNA
HeLa cells were sown on 96-well plates and transfected with PDE3A and non-target siRNA smart pools (On Target Plus, Thermo Scientific) after 24 hours as recommended by the manufacturer. HeLa cytolysis was obtained 24 and 72 hours after transfection for PDE3A and actin (1: 20000, Cell Signaling) as described in the linker-affinity purification and immunoblot of the molecular target of DNMDP. Immunoblot was performed. HeLa cells were treated with compound 3 at the indicated concentration for 48 hours. Cell viability was assessed as described in Compound Library Screening for NCI-H1734 and A549 cell lines.

Measurement of cellular cAMP concentration in HeLa cells
5000 HeLa cells were sown on a 96-well plate. Twenty-four hours after plating, HeLa cells were incubated for 1 hour with the indicated compounds at the indicated concentrations. cAMP levels were measured using the CAMP-GLO ™ assay (Promega) according to the manufacturer's recommendations. Cell concentration of cAMP was determined by normalizing to a standard curve created according to the manufacturer's recommendations.

Extended proteomics method for PDE3A-protein interaction studies
Immunoprecipitation of PDE3A in HeLa cells
HeLa cells were treated with 10 μM of the indicated compounds: DMSO, DNMDP and trekincin for 4 hours prior to lysis. ModRipa lysis buffer (1% NP-40) supplemented with protease and phosphatase inhibitors, and the above indicated compounds up to a final concentration of 10 μM, as described in the linker-affinity purification and immunoblot of the molecular target of DNMDP. HeLa cells were lysed with 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.1% sodium deoxycholate, 1 mM EDTA). 13 mg of HeLa whole cell lysate was incubated with 0.5% PDE3A antibody (Bethyl) and incubated overnight. A blocking peptide (Bethyl) to the PDE3A antibody was added simultaneously with the PDE3A antibody under the corresponding conditions. The whole cell lysate and antibody mixture were then incubated with 10 μl of Protein A plus agarose (Fisher Scientific) at 4 ° C. for 30 minutes. Protein A plus agarose was then washed twice with lysis buffer containing the indicated compound at a concentration of 10 μM. Finally, Protein A plus agarose was washed once with lysis buffer without NP-40 and with the indicated compound at a concentration of 10 μM.

On-bead digestion
The beads from immunopurification were washed once with IP lysis buffer, then three times with PBS, and the three different lysates of each iteration were resuspended in 90 μL digestion buffer (2M urea, 50 mM Tris HCl). , 2 μg of sequencing grade trypsin was added and shaken at 700 rpm for 1 hour. The supernatant was removed and placed in a new tube. The beads were then washed twice with 50 μL of digestion buffer and combined with the supernatant. The combined supernatant was reduced (2 μL 500 mM DTT, 30 min, room temperature), alkylated (4 μL 500 mM IAA, 45 min, dark) and subjected to longer overnight digestion: trypsin 2 μg (4 μL), shake overnight. The sample was then quenched with 20 uL of 10% folic acid (FA) and desalted on a 10 mg SEP-PAK® column.

ITRAQ Labeling of Peptides and Strong Cation Exchange (scx) Fractions Isobarized peptides for relative and absolute quantification (iTRAQ) -reagents according to the manufacturer's instructions (AB Sciex, Foster City, CA). Marked with. The peptide was dissolved in 30 μl of 0.5 M TEAB pH 8.5 solution and the labeling reagent was added to 70 μl of ethanol. After 1 hour of incubation, the reaction was stopped at 50 mM Tris / HCl pH 7.5. The differentially labeled peptides were mixed and then desalted on a 10 mg SEP-PAK® column.

The SCX fraction of the differentially labeled and bound peptide was subjected to 6 pH steps (buffers) as described in Rappsilber et al. (Rappsilber et al., Nat Protoc 2, 1896-1906, 2007). Liquid-all containing 25% acetonitrile):
1: Ammonium acetate 50 mM, pH 4.5,
2: Ammonium acetate 50 mM pH 5.5,
3: Ammonium acetate 50 mM, pH 6.5,
4: Ammonium bicarbonate 50mM pH8,
5: Ammonia hydroxide 0.1% pH 9,
6: Ammonium hydroxide 0.1% pH 11.

As described in this paper, stop-and-go extraction chips (StageTips) were created using Empore SCX discs.

MS Analysis Reconstituted peptides were separated on an online nanoflow EASY-NLC ™ 1000UHPLC system (Thermo Fisher Scientific) and analyzed on a desktop Orbitrap Q EXACTIVE ™ mass spectrometer (Thermo Fisher Scientific). A capillary column (10 μm tip opening / 75 μm diameter) in-house filled with peptide samples using 20 cm C18 silica material (1.9 μm REPROSIL-PUR® C18-AQ medium, Dr. Maisch GmbH, r119.aq). It was injected into PICOFRIT®, New Objective, PF360-75-10-N-5). The UHPLC setup was connected to a custom fit micro-compatible tee (360 μm, IDEX Health & Science, UH-753) and the capillary column was heated to 50 ° C. with a column heater sleeve (Phoenix-ST) to reduce back pressure during UHPLC separation. .. The injected peptide is subjected to a linear 80-minute gradient from 100% solvent A (3% acetonitrile, 0.1% formic acid) to 30% solvent B (90% acetonitrile, 0.1% formic acid), followed by 30% solvent B to 90. Separation was performed with a linear 6-minute gradient of% solvent B at a flow rate of 200 nL / min. Each sample was run for 120 minutes including sample loading and column equilibration time. Q EXACTIVE ™ device with high energy after each MS1 scan (R = 70000) with the 12 top most abundant ions using 3 × 106 ion MS1 ion target and 5 × 104 ion MS2 target. It was operated in data-dependent mode to acquire collision dissociation (HCD) MS / MS scan (R = 17500). The maximum ion time used for MS / MS scans was 120 ms; the HCD normalized collision energy was set to 27; the dynamic exclusion time was set to 20 seconds, and peptide matching and isotope exclusion functions were enabled. bottom.

Peptide and Protein Quantification and Identification Spectrum Mill software package v4.1 beta containing modules developed by Applicants for isobaric tags for quantification based on relative and absolute quantification (iTRAQ) for all mass spectra. Processed using (Agilent Technologies). Precursor ion quantification was performed using an extracted ion chromatogram (XIC's) for each precursor ion. Liquid chromatography (LC) -MS / MS performance of the XIC peak area of each precursor ion subjected to MS / MS using narrow windows around each individual member of the isotope cluster by Spectrum Mill software. Calculated automatically by high resolution MS1 scan intervening. Dynamically determine peak widths in both time and m / z regions based on MS scan resolution, precursor charge and m / z, subject to quality metrics pair theory of relative distribution of peaks in isotope clusters. bottom. Similar MS / MS spectra obtained with the same precursor m / z in the same dissociation mode were merged within +/- 60 seconds. Precursor charge> 7 and poor quality MS / MS spectra that failed the quality filter by not having sequence tag length> 1 (ie, the minimum of 3 masses separated by the intrachain mass of amino acids). MS / MS spectra having were excluded from the search.

For peptide identification, MS / MS spectra were searched against a human universal protein resource (Uniprot) database attached with a common set of laboratory contaminating proteins. Search parameters include ESI-Q EXACTIVE ™ -HCD scoring parameters, trypsin enzyme specificity with up to two missing cleavages, 40% minimum matching peak intensity, +/- 20 ppm precursor mass tolerance, +/ Includes -20 ppm product amount tolerance, carbamide methylation of cysteine as an immobilization modification, and iTRAQ labeling at the lysine and peptide n-terminus. Acceptable variable modifications are methionine, N-terminal acetylation, pyroglutamic acid (N-terminal Q), deamidation (N), pyrocarbamidemethylCys (N-termC), precursor MH + shift range -18 to 64 Da. there were. Automatically interpret the identity for individual spectra by optimizing the scores and delta rank 1 to rank 2 score thresholds separately for each precursor charge state in each liquid chromatography (LC) -MS / MS. It was shown to be effective and enabled a maximum target-decoy-based false positive rate (FDR) of 1.0% at the spectral level.

In calculating the score at the protein level and reporting the identified protein, the redundancy is treated as follows: The protein score is the sum of the scores of different peptides. A distinct peptide is an example of a single highest score for a peptide detected through the MS / MS spectrum. MS / MS spectra for a particular peptide may have been recorded multiple times (ie, isolated from adjacent SCX fractions as different precursor charge states and modified by oxidation of Met), but still It counts as a single, distinct peptide. If multiple protein entries in the sequence database contain peptide sequences of greater than 8 residue lengths, group the proteins and report the highest score 1 and its accession number. When protein sequences are grouped in this way, there may be distinct peptides that uniquely represent the lower scored members (isoforms or family members) of the group. Each of these examples spawns a subgroup, multiple subgroups are reported and counted towards the total number of proteins. The iTRAQ ratio was obtained from the Spectrum Mill protein comparison export table. Median values were calculated across all the different peptides assigned to each repeat protein subgroup to obtain the iTRAQ protein ratio. To assign interacting proteins, use the Limma package of the R environment to calculate the relaxed t-test p as previously described and exclude proteins with a reproducibility of less than 95% Blandt -Added Altman test (Udeshi et al., Mol Cell Proteomics 11, 148-159, 2012).

Verification of DNMDP-induced PDE3A protein interactions using immunoprecipitation and immunoblot
HeLa cells were transfected with an ORF overexpressing construct expressing V5-tagged SIRT7, V5-tagged SLFN12 or V5-tagged GFP. The ORF expression construct was obtained from TRC (clone ID: TRCN0000468231, TRCN0000476272, ccsbBroad304_99997). After 72 hours of transfection, cells were treated with 10 μM DNMDP or trekincin for 4 hours, followed by lysis with ModRipa lysis buffer to immunoprecipitate PDE3A. For each condition, 2 mg of total protein lysate was incubated overnight with 1 μg of anti-PDE3A antibody at 4 ° C, then 7.5 μl of protein A- and protein G-Dynabeads, respectively (Life Technologies 10001D and 10003D) were added, followed by an additional 1 Incubated for hours. The beads were washed and the bound protein was eluted with 30 μl of LDS PAGE gel loading buffer. Separation of input (total protein lysate approx. 60 μg) and IP product on 4-12% Tris-glycine PAGE gel, anti-V5 antibody (Life Technologies R96205, 1: 5000), Bethyl anti-PDE3A antibody (1: 1000). And secondary antibodies from LiCOR Biosciences (Cat. Nos. 926-3210 and 926068021, 1: 10000 respectively) were immunoblotted. The blots were washed and imaged using a LiCOR Odyssey infrared imager.

Knockdown of SLFN12 expression and drug susceptibility testing using shRNA
Constructs expressing shRNA targeting SLFN12, or control vectors, were packaged in lentivirus and delivered to HeLa cells by viral transduction. Three SLFN12 targeted shRNAs were used, all of which were obtained from TRCs (clone IDs: TRCN0000152141 and TRCN0000153520). Infected cells were selected with 1 μg / ml puromycin for 3 days and then grown in non-selective medium for an additional 3 days. The cells were then sown on a 384-well assay plate and tested for drug susceptibility as described above. Knockdown of SLFN12 was verified by qPCR. Total RNA was extracted using the kit reagents (RNeasy Mini Kit (Qiagen # 74104) and QIAschredder (Qiagen # 79656)). CDNA was prepared using the kit reagent (SuperScript III First-Strand Synthesis System (Life Technologies # 18080-051)). qPCR was performed on GAPDH and SLFN12 (Life Technologies Hs00430118_m1) according to the manufacturer's recommendations. SLFN12 expression was normalized to the corresponding sample GAPDH ct value.

Other Embodiments From the above description, it will be clear that modifications and modifications can be made to the invention described herein for adoption in various uses and conditions. Such embodiments are also within the scope of the following claims.

The enumeration of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or partial combination) of enumerated elements. The enumeration of embodiments herein includes the embodiments as any single embodiment or in combination with any other embodiment or a portion thereof.

Built-in by reference ASCII text files submitted here via EFS-Web entitled "167741_011202.txt" created in August 2016 [[TBD]] with the size of [[TBD]] bytes The whole is incorporated herein by reference.

All patents and publications referred to herein are hereby by reference to the same extent that their respective independent patents and publications have been shown to be specifically and individually incorporated by reference. Will be incorporated into. In particular, Lewis et al., "Compounds and Compositions for the Treatment of Cancer", PCT / US2014 / 023263 (Pamphlet of International Publication No. 2014/164704) are incorporated by reference in their entirety.

Claims (18)

An agent for killing or reducing the survival of cancer cells selected as reactive to the phosphodiesterase 3A (PDE3A) modulator, which comprises the PDE3A modulator, wherein the cells are PDE3A and Schlafen12 (SLFN12) relative to the reference. Selected as having an increase in polypeptide or polynucleotide levels, said PDE3A modulator is 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3. (2H) -An agent selected from the group consisting of on (DNMDP) and its pharmaceutically acceptable salts. An agent for reducing cancer cell growth in a subject preselected as having cancer that is responsive to a PDE3A modulator, comprising the PDE3A modulator, wherein the subject is a PDE3A and SLFN12 polypeptide or polynucleotide relative to the reference. Preselected by detecting increased levels, the PDE3A modulator is 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H)-. An agent selected from the group consisting of on (DNMDP) and its pharmaceutically acceptable salts. A method of detecting a subject with cancer cells that is responsive to PDE3A regulation by a PDE3A modulator, detecting an increase in PDE3A and SLFN12 polypeptide or polynucleotide levels in the subject's biological sample relative to the reference, thereby PDE3A. The PDE3A modulator comprises 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-, comprising the step of detecting the subject as having a regulatory responsive cancer. A method selected from the group consisting of 3 (2H) -one (DNMDP) and its pharmaceutically acceptable salts. A method of detecting a subject with cancer resistant to PDE3A regulation by a PDE3A modulator, which detects a decrease in SLFN12 polypeptide or polynucleotide levels in the subject's biological sample relative to the reference, thereby being resistant to PDE3A regulation. Including the step of detecting the subject as having cancer, the PDE3A modulator is 6- (4- (diethylamino) -3-nitrophenyl) -5-methyl-4,5-dihydropyridazine-3 (2H)-. A method selected from the group consisting of on (DNMDP) and its pharmaceutically acceptable salts. The agent according to claim 1 or 2, wherein PDE3A or SLFN12 levels are detected by a method selected from the group consisting of immunoblot, mass spectrometry and immunoprecipitation. The method of claim 3 or 4, wherein PDE3A or SLFN12 levels are detected by a method selected from the group consisting of immunoblot, mass spectrometry and immunoprecipitation. The agent according to claim 1 or 2, wherein PDE3A or SLFN12 polynucleotide levels are detected by a method selected from the group consisting of quantitative PCR, Northern blots, microarrays, mass spectrometry and in situ hybridization. The method of claim 3 or 4, wherein the PDE3A or SLFN12 polynucleotide level is detected by a method selected from the group consisting of quantitative PCR, Northern blots, microarrays, mass spectrometry and in situ hybridization. The cancer cells are melanoma, endometrial cancer, lung cancer, hematopoietic / lymph cancer, ovarian cancer, cervical cancer, soft tissue sarcoma, smooth muscle tumor, urinary tract cancer, pancreatic cancer, and thyroid cancer. The agent according to claim 1 or 2, which is a cancer, kidney cancer, glioblastoma or breast cancer cell. The cancer cells are melanoma, endometrial cancer, lung cancer, hematopoietic / lymph cancer, ovarian cancer, cervical cancer, soft tissue sarcoma, smooth muscle tumor, urinary tract cancer, pancreatic cancer, and thyroid cancer. The method according to claim 3 or 4, which is a cancer, kidney cancer, glioblastoma or breast cancer cell. The agent according to claim 1 or 2, wherein the cancer cells are not B cell proliferative cancers. The method according to claim 3 or 4, wherein the cancer cells are not B cell proliferative cancers. The agent according to claim 1 or 2, wherein the cancer cells are not multiple myeloma. The method of claim 3 or 4, wherein the cancer cells are not multiple myeloma. The agent according to claim 2, which is administered orally or by intravenous injection. The method according to claim 3, wherein the biological sample is a tissue sample containing cancer cells. The agent according to any one of claims 1, 2 and 5, wherein the criteria are health controls. The method of any one of claims 3, 4 and 6, wherein the criteria are health controls.

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